RNA detection assays

ABSTRACT

The present invention provides novel cleavage agents and polymerases for the cleavage and modification of nucleic acid. The cleavage agents and polymerases find use, for example, for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. In some embodiments, the 5′ nuclease activity of a variety of enzymes is used to cleave a target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

[0001] The present application is a continuation-in-part of U.S. patentapplication Ser. No. 09/864,636, and is a continuation-in-part of patentapplication Ser. No. 09/864,426, each of which is acontinutation-in-part of U.S. patent application Ser. No. 09/577,304 and09/758,282 and is a continuation-in-part of U.S. patent application Ser.No. 09/350,309, which is a divisional application U.S. Pat. No.5,985,557, and is also a continuation-in-part of co-pending applicationSer. No. 09/381,212 which is a national entry of PCT application no. US98/05809, which claims priority to U.S. Pat. Nos. 5,994,069, 6,090,543,5,985,557, 6,001,567, and 5,846,717 and PCT application no. US 97/01072.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods for thedetection and characterization of nucleic acid sequences and variationsin nucleic acid sequences. The present invention relates to methods forforming a nucleic acid cleavage structure on a target sequence andcleaving the nucleic acid cleavage structure in a site-specific manner.For example, in some embodiments, the 5′ nuclease activity of a varietyof enzymes is used to cleave the target-dependent cleavage structure,thereby indicating the presence of specific nucleic acid sequences orspecific variations thereof.

BACKGROUND OF THE INVENTION

[0003] Methods for the detection and characterization of specificnucleic acid sequences and sequence variations have been used to detectthe presence of viral or bacterial nucleic acid sequences indicative ofan infection, to detect the presence of variants or alleles of genesassociated with disease and cancers. These methods also find applicationin the identification of sources of nucleic acids, as for forensicanalysis or for paternity determinations.

[0004] Various methods are known to the art that may be used to detectand characterize specific nucleic acid sequences and sequence variants.Nonetheless, with the completion of the nucleic acid sequencing of thehuman genome, as well as the genomes of numerous pathogenic organisms,the demand for fast, reliable, cost-effective and user-friendly testsfor the detection of specific nucleic acid sequences continues to grow.Importantly, these tests must be able to create a detectable signal fromsamples that contain very few copies of the sequence of interest. Thefollowing discussion examines two levels of nucleic acid detectionassays currently in use: I. Signal Amplification Technology fordetection of rare sequences II. Direct Detection Technology forquantitative detection of sequences, and III. Direct Detection of RNA.

[0005] I. Signal Amplification Technology Methods for Amplification

[0006] The “Polymerase Chain Reaction” (PCR) comprises the firstgeneration of methods for nucleic acid amplification. However, severalother methods have been developed that employ the same basis ofspecificity, but create signal by different amplification mechanisms.These methods include the “Ligase Chain Reaction” (LCR), “Self-SustainedSynthetic Reaction” (3SR/NASBA), and “Qβ-Replicase” (Qβ).

[0007] Polymerase Chain Reaction (PCR)

[0008] The polymerase chain reaction (PCR), as described in U.S. Pat.Nos. 4,683,195, 4,683,202, and 4,965,188 to Mullis and Mullis et al.(the disclosures of which are hereby incorporated by reference),describe a method for increasing the concentration of a segment oftarget sequence in a mixture of genomic DNA without cloning orpurification. This technology provides one approach to the problems oflow target sequence concentration. PCR can be used to directly increasethe concentration of the target to an easily detectable level. Thisprocess for amplifying the target sequence involves introducing a molarexcess of two oligonucleotide primers that are complementary to theirrespective strands of the double-stranded target sequence to the DNAmixture containing the desired target sequence. The mixture is denaturedand then allowed to hybridize. Following hybridization, the primers areextended with polymerase so as to form complementary strands. The stepsof denaturation, hybridization, and polymerase extension can be repeatedas often as needed, in order to obtain relatively high concentrations ofa segment of the desired target sequence.

[0009] The length of the segment of the desired target sequence isdetermined by the relative positions of the primers with respect to eachother, and, therefore, this length is a controllable parameter. Becausethe desired segments of the target sequence become the dominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR-amplified.”

[0010] Ligase Chain Reaction (LCR or LAR)

[0011] The ligase chain reaction (LCR; sometimes referred to as “LigaseAmplification Reaction” (LAR) described by Barany, Proc. Natl. Acad.Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wuand Wallace, Genomics 4:560 (1989) has developed into a well-recognizedalternative method for amplifying nucleic acids. In LCR, fouroligonucleotides, two adjacent oligonucleotides that uniquely hybridizeto one strand of target DNA, and a complementary set of adjacentoligonucleotides, that hybridize to the opposite strand are mixed andDNA ligase is added to the mixture. Provided that there is completecomplementarity at the junction, ligase will covalently link each set ofhybridized molecules. Importantly, in LCR, two probes are ligatedtogether only when they base-pair with sequences in the target sample,without gaps or mismatches. Repeated cycles of denaturation,hybridization and ligation amplify a short segment of DNA. LCR has alsobeen used in combination with PCR to achieve enhanced detection ofsingle-base changes. Segev, PCT Public. No. WO9001069 A1 (1990).However, because the four oligonucleotides used in this assay can pairto form two short ligatable fragments, there is the potential for thegeneration of target-independent background signal. The use of LCR formutant screening is limited to the examination of specific nucleic acidpositions.

[0012] Self-Sustained Synthetic Reaction (3SR/NASBA) The self-sustainedsequence replication reaction (3SR) (Guatelli et al., Proc. Natl. Acad.Sci., 87:1874-1878 [1990], with an erratum at Proc. Natl. Acad. Sci.,87:7797 [1990]) is a transcription-based in vitro amplification system(Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177 [1989]) that canexponentially amplify RNA sequences at a uniform temperature. Theamplified RNA can then be utilized for mutation detection (Fahy et al.,PCR Meth. Appl., 1:25 [1991]). In this method, an oligonucleotide primeris used to add a phage RNA polymerase promoter to the 5′ end of thesequence of interest. In a cocktail of enzymes and substrates thatincludes a second primer, reverse transcriptase, RNase H, RNA polymeraseand ribo-and deoxyribonucleoside triphosphates, the target sequenceundergoes repeated rounds of transcription, cDNA synthesis andsecond-strand synthesis to amplify the area of interest. The use of 3SRto detect mutations is kinetically limited to screening small segmentsof DNA (e.g., 200-300 base pairs).

[0013] Q-Beta (Qβ) Replicase

[0014] In this method, a probe that recognizes the sequence of interestis attached to the replicatable RNA template for Qβ replicase. Apreviously identified major problem with false positives resulting fromthe replication of unhybridized probes has been addressed through use ofa sequence-specific ligation step. However, available thermostable DNAligases are not effective on this RNA substrate, so the ligation must beperformed by T4 DNA ligase at low temperatures (37° C.). This preventsthe use of high temperature as a means of achieving specificity as inthe LCR, the ligation event can be used to detect a mutation at thejunction site, but not elsewhere.

[0015] Table 2 below, lists some of the features desirable for systemsuseful in sensitive nucleic acid diagnostics, and summarizes theabilities of each of the major amplification methods (See also,Landgren, Trends in Genetics 9:199 [1993]).

[0016] A successful diagnostic method must be very specific. Astraight-forward method of controlling the specificity of nucleic acidhybridization is by controlling the temperature of the reaction. Whilethe 3SR/NASBA, and Qβ systems are all able to generate a large quantityof signal, one or more of the enzymes involved in each cannot be used athigh temperature (i.e., >55° C.). Therefore the reaction temperaturescannot be raised to prevent non-specific hybridization of the probes. Ifprobes are shortened in order to make them melt more easily at lowtemperatures, the likelihood of having more than one perfect match in acomplex genome increases. For these reasons, PCR and LCR currentlydominate the research field in detection technologies. TABLE 1 MethodPCR & 3SR Feature PCR LCR LCR NASBA Qβ Amplifies Target + + + +Recognition of Independent + + + + + Sequences Required Performed atHigh Temp. + + Operates at Fixed Temp. + + ExponentialAmplification + + + + + Generic Signal Generation + Easily Automatable

[0017] The basis of the amplification procedure in the PCR and LCR isthe fact that the products of one cycle become usable templates in allsubsequent cycles, consequently doubling the population with each cycle.The final yield of any such doubling system can be expressed as:(1+X)^(n)=y, where “X” is the mean efficiency (percent copied in eachcycle), “n” is the number of cycles, and “y” is the overall efficiency,or yield of the reaction (Mullis, PCR Methods Applic., 1:1 [1991]). Ifevery copy of a target DNA is utilized as a template in every cycle of apolymerase chain reaction, then the mean efficiency is 100%. If 20cycles of PCR are performed, then the yield will be 220, or 1,048,576copies of the starting material. If the reaction conditions reduce themean efficiency to 85%, then the yield in those 20 cycles will be only1.85²⁰, or 220,513 copies of the starting material. In other words, aPCR running at 85% efficiency will yield only 21% as much final product,compared to a reaction running at 100% efficiency. A reaction that isreduced to 50% mean efficiency will yield less than 1% of the possibleproduct.

[0018] In practice, routine polymerase chain reactions rarely achievethe theoretical maximum yield, and PCRs are usually run for more than 20cycles to compensate for the lower yield. At 50% mean efficiency, itwould take 34 cycles to achieve the million-fold amplificationtheoretically possible in 20, and at lower efficiencies, the number ofcycles required becomes prohibitive. In addition, any backgroundproducts that amplify with a better mean efficiency than the intendedtarget will become the dominant products.

[0019] Also, many variables can influence the mean efficiency of PCR,including target DNA length and secondary structure, primer length anddesign, primer and dNTP concentrations, and buffer composition, to namebut a few. Contamination of the reaction with exogenous DNA (e.g., DNAspilled onto lab surfaces) or cross-contamination is also a majorconsideration. Reaction conditions must be carefully optimized for eachdifferent primer pair and target sequence, and the process can takedays, even for an experienced investigator. The laboriousness of thisprocess, including numerous technical considerations and other factors,presents a significant drawback to using PCR in the clinical setting.Indeed, PCR has yet to penetrate the clinical market in a significantway. The same concerns arise with LCR, as LCR must also be optimized touse different oligonucleotide sequences for each target sequence. Inaddition, both methods require expensive equipment, capable of precisetemperature cycling.

[0020] Many applications of nucleic acid detection technologies, such asin studies of allelic variation, involve not only detection of aspecific sequence in a complex background, but also the discriminationbetween sequences with few, or single, nucleotide differences. Onemethod for the detection of allele-specific variants by PCR is basedupon the fact that it is difficult for Taq polymerase to synthesize aDNA strand when there is a mismatch between the template strand and the3′ end of the primer. An allele-specific variant may be detected by theuse of a primer that is perfectly matched with only one of the possiblealleles; the mismatch to the other allele acts to prevent the extensionof the primer, thereby preventing the amplification of that sequence.This method has a substantial limitation in that the base composition ofthe mismatch influences the ability to prevent extension across themismatch, and certain mismatches do not prevent extension or have only aminimal effect (Kwok et al, Nucl. Acids Res., 18:999 [1990]).)

[0021] A similar 3′-mismatch strategy is used with greater effect toprevent ligation in the LCR (Barany, PCR Meth. Applic., 1:5 [1991]). Anymismatch effectively blocks the action of the thermostable ligase, butLCR still has the drawback of target-independent background ligationproducts initiating the amplification. Moreover, the combination of PCRwith subsequent LCR to identify the nucleotides at individual positionsis also a clearly cumbersome proposition for the clinical laboratory.

[0022] II. Direct Detection Technology

[0023] When a sufficient amount of a nucleic acid to be detected isavailable, there are advantages to detecting that sequence directly,instead of making more copies of that target, (e.g., as in PCR and LCR).Most notably, a method that does not amplify the signal exponentially ismore amenable to quantitative analysis. Even if the signal is enhancedby attaching multiple dyes to a single oligonucleotide, the correlationbetween the final signal intensity and amount of target is direct. Sucha system has an additional advantage that the products of the reactionwill not themselves promote further reaction, so contamination of labsurfaces by the products is not as much of a concern. Traditionalmethods of direct detection including Northern and Southern blotting andRNase protection assays usually require the use of radioactivity and arenot amenable to automation. Recently devised techniques have sought toeliminate the use of radioactivity and/or improve the sensitivity inautomatable formats. Two examples are the “Cycling Probe Reaction”(CPR), and “Branched DNA” (bDNA)

[0024] The cycling probe reaction (CPR) (Duck et al., BioTech., 9:142[1990]), uses a long chimeric oligonucleotide in which a central portionis made of RNA while the two termini are made of DNA. Hybridization ofthe probe to a target DNA and exposure to a thermostable RNase H causesthe RNA portion to be digested. This destabilizes the remaining DNAportions of the duplex, releasing the remainder of the probe from thetarget DNA and allowing another probe molecule to repeat the process.The signal, in the form of cleaved probe molecules, accumulates at alinear rate. While the repeating process increases the signal, the RNAportion of the oligonucleotide is vulnerable to RNases that may becarried through sample preparation.

[0025] Branched DNA (bDNA), described by Urdea et al., Gene 61:253-264(1987), involves oligonucleotides with branched structures that alloweach individual oligonucleotide to carry 35 to 40 labels (e.g., alkalinephosphatase enzymes). While this enhances the signal from ahybridization event, signal from non-specific binding is similarlyincreased.

[0026] While both of these methods have the advantages of directdetection discussed above, neither the CPR or bDNA methods can make useof the specificity allowed by the requirement of independent recognitionby two or more probe (oligonucleotide) sequences, as is common in thesignal amplification methods described in Section I. above. Therequirement that two oligonucleotides must hybridize to a target nucleicacid in order for a detectable signal to be generated confers an extrameasure of stringency on any detection assay. Requiring twooligonucleotides to bind to a target nucleic acid reduces the chancethat false “positive” results will be produced due to the non-specificbinding of a probe to the target. The further requirement that the twooligonucleotides must bind in a specific orientation relative to thetarget, as is required in PCR, where oligonucleotides must be oppositelybut appropriately oriented such that the DNA polymerase can bridge thegap between the two oligonucleotides in both directions, furtherenhances specificity of the detection reaction. However, it is wellknown to those in the art that even though PCR utilizes twooligonucleotide probes (termed primers) “non-specific” amplification(i.e., amplification of sequences not directed by the two primers used)is a common artifact. This is in part because the DNA polymerase used inPCR can accommodate very large distances, measured in nucleotides,between the oligonucleotides and thus there is a large window in whichnon-specific binding of an oligonucleotide can lead to exponentialamplification of inappropriate product. The LCR, in contrast, cannotproceed unless the oligonucleotides used are bound to the targetadjacent to each other and so the full benefit of the dualoligonucleotide hybridization is realized.

[0027] An ideal direct detection method would combine the advantages ofthe direct detection assays (e.g., easy quantification and minimal riskof carry-over contamination) with the specificity provided by a dualoligonucleotide hybridization assay.

[0028] III. Direct Detection of RNA

[0029] In molecular medicine, a simple and cost-effective method fordirect and quantitative RNA detection would greatly facilitate theanalysis of RNA viruses and the measurement of specific gene expression.Both of these issues are currently pressing problems in the field.Despite this need, few techniques have emerged that are truly direct.PCR-based detection assays require conversion of RNA to DNA by reversetranscriptase before amplification, introducing a variable that cancompromise accurate quantification. Furthermore, PCR and other methodsbased on exponential amplification (e.g., NASBA) require painstakingcontainment measures to avoid cross-contamination, and have difficultydistinguishing small differences (e.g., 2 to 3-fold) in quantity. Othertests that directly examine RNA suffer from a variety of drawbacks,including time consuming autoradiography steps (e.g., RNase protectionassays), or overnight reaction times (e.g., branched DNA assays). Withover 1.5 million viral load measurements being performed in the U.S.every year, there is clearly an enormous potential for an inexpensive,rapid, high-throughput system for the quantitative measurement of RNA.

[0030] Techniques for direct, quantitative detection of mRNA are vitalfor monitoring expression of a number of different genes. In particular,levels of cytokine expression (e.g., interleukins and lymphokines) arebeing exploited as clinical measures of immune response in theprogression of a wide variety of diseases (Van Deuren et al., J. Int.Fed. Clin. Chem., 5:216 [1993], Van Deuren et al., J. Inf. Dis., 169:157[1994], Perenboom et al., Eur. J. Clin. Invest., 26:159 [1996], Guidottiet al., Immunity 4:25 [1996]) as well as in monitoring transplantrecipients (Grant et al., Transplantation 62:910 [1996]). Additionally,the monitoring of viral load and identification of viral genotype havegreat clinical significance for individuals suffering viral infectionsby such pathogens as HIV or Hepatitis C virus (HCV). There is a highcorrelation between viral load (i.e., the absolute number of viralparticles in the bloodstream) and time to progression to AIDS (Mellorset al., Science 272:1167 [1996], Saag et al., Nature Medicine 2:625[1996]). For that reason, viral load, as measured by quantitativenucleic acid based testing, is becoming a standard monitoring procedurefor evaluating the efficacy of treatment and the clinical status of HIVpositive patients. It is thought to be essential to reduce viral load asearly in the course of infection as possible and to evaluate virallevels on a regular basis. In the case of HCV, viral genotype has greatclinical significance, with correlations to severity of liver diseaseand responsiveness to interferon therapy. Furthermore, because HCVcannot be grown in culture, it is only by establishing correlationsbetween characteristics like viral genotype and clinical outcome thatnew antiviral treatments can be evaluated.

[0031] While the above mentioned methods have been serviceable for lowthroughput, research applications, or for limited clinical application,it is clear that large scale quantitative analysis of RNA readilyadaptable to any genetic system will require a more innovative approach.An ideal direct detection method would combine the advantages of thedirect detection assays (e.g., easy quantification and minimal risk ofcarry-over contamination) with the specificity provided by a dualoligonucleotide hybridization assay.

[0032] Many of the methods described above rely on hybridization aloneto distinguish a target molecule from other nucleic acids. Although someof these methods can be highly sensitive, they often cannot quantitateand distinguish closely related mRNAs accurately, especially such RNAsexpressed at different levels in the same sample. While theabove-mentioned methods are serviceable for some purposes, a need existsfor a technology that is particularly adept at distinguishing particularRNAs from closely related molecules.

SUMMARY OF THE INVENTION

[0033] The present invention relates to compositions and methods for thedetection and characterization of nucleic acid sequences and variationsin nucleic acid sequences. The present invention relates to methods forforming a nucleic acid cleavage structure on a target sequence andcleaving the nucleic acid cleavage structure in a site-specific manner.For example, in some embodiments, the 5′ nuclease activity of a varietyof enzymes is used to cleave the target-dependent cleavage structure,thereby indicating the presence of specific nucleic acid sequences orspecific variations thereof.

[0034] The present invention provides structure-specific cleavage agents(e.g., nucleases) from a variety of sources, including mesophilic,psychrophilic, thermophilic, and hyperthermophilic organisms. Thepreferred structure-specific nucleases are thermostable. Thermostablestructure-specific nucleases are contemplated as particularly useful inthat they operate at temperatures where nucleic acid hybridization isextremely specific, allowing for allele-specific detection (includingsingle-base mismatches). In one embodiment, the thermostablestructure-specific nucleases are thermostable 5′ nucleases comprisingaltered polymerases derived from the native polymerases of Thermusspecies, including, but not limited to Thermus aquaticus, Thermusflavus, and Thermus thermophilus. However, the invention is not limitedto the use of thermostable 5′ nucleases. Thermostable structure-specificnucleases from the FEN-1, RAD2 and XPG class of nucleases are alsopreferred.

[0035] The present invention provides a method for detecting a targetsequence (e.g., a mutation, polymorphism, etc), comprising providing asample suspected of containing the target sequence; oligonucleotidescapable of forming an invasive cleavage structure in the presence of thetarget sequence; and an agent for detecting the presence of an invasivecleavage structure; and exposing the sample to the oligonucleotides andthe agent. In some embodiments, the method further comprises the step ofdetecting a complex comprising the agent and the invasive cleavagestructure (directly or indirectly). In some embodiments, the agentcomprises a cleavage agent. In some preferred embodiments, the exposingof the sample to the oligonucleotides and the agent comprises exposingthe sample to the oligonucleotides and the agent under conditionswherein an invasive cleavage structure is formed between the targetsequence and the oligonucleotides if the target sequence is present inthe sample, wherein the invasive cleavage structure is cleaved by thecleavage agent to form a cleavage product. In some embodiments, themethod further comprises the step of detecting the cleavage product. Insome embodiments, the target sequence comprises a first region and asecond region, the second region downstream of and contiguous to thefirst region, and wherein the oligonucleotides comprise first and secondoligonucleotides, wherein at least a portion of the firstoligonucleotide is completely complementary to the first portion of thetarget sequence and wherein the second oligonucleotide comprises a 3′portion and a 5′ portion, wherein the 5′ portion is completelycomplementary to the second portion of said target nucleic acid.

[0036] The present invention also provides a kit for detecting suchtarget sequences, said kit comprising oligonucleotides capable offorming an invasive cleavage structure in the presence of the targetsequence. In some embodiments, the kit further comprises an agent fordetecting the presence of an invasive cleavage structure (e.g., acleavage agent). In some embodiments, the oligonucleotides comprisefirst and second oligonucleotides, said first oligonucleotide comprisinga 5′ portion complementary to a first region of the target nucleic acidand said second oligonucleotide comprising a 3′ portion and a 5′portion, said 5′ portion complementary to a second region of the targetnucleic acid downstream of and contiguous to the first portion. In somepreferred embodiments, the target sequence comprises

[0037] The present invention also provides methods for detecting thepresence of a target nucleic acid molecule by detecting non-targetcleavage products comprising providing: a cleavage agent; a source oftarget nucleic acid, the target nucleic acid comprising a first regionand a second region, the second region downstream of and contiguous tothe first region; a first oligonucleotide, wherein at least a portion ofthe first oligonucleotide is completely complementary to the firstportion of the target nucleic acid; and a second oligonucleotidecomprising a 3′ portion and a 5′ portion, wherein the 5′ portion iscompletely complementary to the second portion of the target nucleicacid; mixing the cleavage agent, the target nucleic acid, the firstoligonucleotide and the second oligonucleotide to create a reactionmixture under reaction conditions such that at least the portion of thefirst oligonucleotide is annealed to the first region of said targetnucleic acid and wherein at least the 5′ portion of the secondoligonucleotide is annealed to the second region of the target nucleicacid so as to create a cleavage structure, and wherein cleavage of thecleavage structure occurs to generate non-target cleavage product; anddetecting the cleavage of the cleavage structure.

[0038] The detection of the cleavage of the cleavage structure can becarried out in any manner. In some embodiments, the detection of thecleavage of the cleavage structure comprises detecting the non-targetcleavage product. In yet other embodiments, the detection of thecleavage of the cleavage structure comprises detection of fluorescence,mass, or fluorescence energy transfer. Other detection methods include,but are not limited to detection of radioactivity, luminescence,phosphorescence, fluorescence polarization, and charge. In someembodiments, detection is carried out by a method comprising providingthe non-target cleavage product; a composition comprising twosingle-stranded nucleic acids annealed so as to define a single-strandedportion of a protein binding region; and a protein; and exposing thenon-target cleavage product to the single-stranded portion of theprotein binding region under conditions such that the protein binds tothe protein binding region. In some embodiments, the protein comprises anucleic acid producing protein, wherein the nucleic acid producingprotein binds to the protein-binding region and produces nucleic acid.In some embodiments, the protein-binding region is a template-dependentRNA polymerase binding region (e.g., a T7 RNA polymerase bindingregion). In other embodiments, the detection is carried out by a methodcomprising providing the non-target cleavage product; a singlecontinuous strand of nucleic acid comprising a sequence defining asingle strand of an RNA polymerase binding region; a template-dependentDNA polymerase; and a template-dependent RNA polymerase; exposing thenon-target cleavage product to the RNA polymerase binding region underconditions such that the non-target cleavage product binds to a portionof the single strand of the RNA polymerase binding region to produce abound non-target cleavage product; exposing the bound non-targetcleavage product to the template-dependent DNA polymerase underconditions such that a double-stranded RNA polymerase binding region isproduced; and exposing the double-stranded RNA polymerase binding regionto the template-dependent RNA polymerase under conditions such that RNAtranscripts are produced. In some embodiments, the method furthercomprises the step of detecting the RNA transcripts. In someembodiments, the template-dependent RNA polymerase is T7 RNA polymerase.

[0039] The present invention is not limited by the nature of the 3′portion of the second oligonucleotide. In some preferred embodiments,the 3′ portion of the second oligonucleotide comprises a 3′ terminalnucleotide not complementary to the target nucleic acid. In someembodiments, the 3′ portion of the second oligonucleotide consists of asingle nucleotide not complementary to the target nucleic acid.

[0040] Any of the components of the method may be attached to a solidsupport. For example, in some embodiments, the first oligonucleotide isattached to a solid support. In other embodiments, the secondoligonucleotide is attached to a solid support.

[0041] The cleavage agent can be any agent that is capable of cleavinginvasive cleavage structures. In some preferred embodiments, thecleavage agent comprises a structure-specific nuclease. In particularlypreferred embodiments, the structure-specific nuclease comprises athermostable structure-specific nuclease (e.g., a thermostable 5′nuclease). Thermostable structure-specific nucleases include, but arenot limited to, those having an amino acid sequence homologous to aportion of the amino acid sequence of a thermostable DNA polymerasederived from a thermophilic organism (e.g., Thermus aquaticus, Thermusflavus, and Thermus thermophilus). In other embodiments, thethermostable structure-specific nuclease comprises a nuclease from theFEN-1, RAD2 or XPG classes of nucleases, or chimerical structurescontaining one or more portions of any of the above cleavage agents.

[0042] The method is not limited by the nature of the target nucleicacid. In some embodiments, the target nucleic acid is single stranded ordouble stranded DNA or RNA. In some embodiments, double stranded nucleicacid is rendered single stranded (e.g., by heat) prior to formation ofthe cleavage structure. In some embodiment, the source of target nucleicacid comprises a sample containing genomic DNA. Sample include, but arenot limited to, blood, saliva, cerebral spinal fluid, pleural fluid,milk, lymph, sputum and semen.

[0043] In some embodiments, the reaction conditions for the methodcomprise providing a source of divalent cations. In some preferredembodiments, the divalent cation is selected from the group comprisingMn²⁺ and Mg²⁺ ions. In some embodiments, the reaction conditions for themethod comprise providing the first and the second oligonucleotides inconcentration excess compared to the target nucleic acid.

[0044] In some embodiments, the method further comprises providing athird oligonucleotide complementary to a third portion of said targetnucleic acid upstream of the first portion of the target nucleic acid,wherein the third oligonucleotide is mixed with the reaction mixture.

[0045] The present invention also provides a method for detecting thepresence of a target nucleic acid molecule by detecting non-targetcleavage products comprising providing: a cleavage agent; a source oftarget nucleic acid, the target nucleic acid comprising a first regionand a second region, the second region downstream of and contiguous tothe first region; a plurality of first oligonucleotides, wherein atleast a portion of the first oligonucleotides is completelycomplementary to the first portion of the target nucleic acid; a secondoligonucleotide comprising a 3′ portion and a 5′ portion, wherein said5′ portion is completely complementary to the second portion of thetarget nucleic acid; mixing the cleavage agent, the target nucleic acid,the plurality of first oligonucleotides and second oligonucleotide tocreate a reaction mixture under reaction conditions such that at leastthe portion of a first oligonucleotide is annealed to the first regionof the target nucleic acid and wherein at least the 5′ portion of thesecond oligonucleotide is annealed to the second region of the targetnucleic acid so as to create a cleavage structure, and wherein cleavageof the cleavage structure occurs to generate non-target cleavageproduct, wherein the conditions permit multiple cleavage structures toform and be cleaved from the target nucleic acid; and detecting thecleavage of said cleavage structures. In some embodiments, theconditions comprise isothermal conditions that permit the plurality offirst oligonucleotides to dissociate from the target nucleic acid. Whilethe present invention is limited by the number of cleavage structureformed on a particular target nucleic acid, in some preferredembodiments, two or more (3, 4, 5, . . . , 10, . . . , 10000, . . . ) ofthe plurality of first oligonucleotides form cleavage structures with aparticular target nucleic acid, wherein the cleavage structures arecleaved to produce the non-target cleavage products.

[0046] The present invention also provides methods wherein a cleavageproduct from the above methods is used in a further invasive cleavagereaction. For example, the present invention provides a methodcomprising providing a cleavage agent; a first target nucleic acid, thefirst target nucleic acid comprising a first region and a second region,the second region downstream of and contiguous to the first region; afirst oligonucleotide, wherein at least a portion of the firstoligonucleotide is completely complementary to the first portion of thefirst target nucleic acid; a second oligonucleotide comprising a 3′portion and a 5′ portion, wherein the 5′ portion is completelycomplementary to the second portion of the first target nucleic acid; asecond target nucleic acid, said second target nucleic acid comprising afirst region and a second region, the second region downstream of andcontiguous to the first region; and a third oligonucleotide, wherein atleast a portion of the third oligonucleotide is completely complementaryto the first portion of the second target nucleic acid; generating afirst cleavage structure wherein at least said portion of the firstoligonucleotide is annealed to the first region of the first targetnucleic acid and wherein at least the 5′ portion of the secondoligonucleotide is annealed to the second region of the first targetnucleic acid and wherein cleavage of the first cleavage structure occursvia the cleavage agent thereby cleaving the first oligonucleotide togenerate a fourth oligonucleotide, said fourth oligonucleotidecomprising a 3′ portion and a 5′ portion, wherein the 5′ portion iscompletely complementary to the second portion of the second targetnucleic acid; generating a second cleavage structure under conditionswherein at least said portion of the third oligonucleotide is annealedto the first region of the second target nucleic acid and wherein atleast the 5′ portion of the fourth oligonucleotide is annealed to thesecond region of the second target nucleic acid and wherein cleavage ofthe second cleavage structure occurs to generate a cleavage fragment;and detecting the cleavage of the second cleavage structure. In somepreferred embodiments, the 3′ portion of the fourth oligonucleotidecomprises a 3′ terminal nucleotide not complementary to the secondtarget nucleic acid. In some embodiments, the 3′ portion of the thirdoligonucleotide is covalently linked to the second target nucleic acid.In some embodiments, the second target nucleic acid further comprises a5′ region, wherein the 5′ region of the second target nucleic acid isthe third oligonucleotide. The present invention further provides kitscomprising: a cleavage agent; a first oligonucleotide comprising a 5′portion complementary to a first region of a target nucleic acid; and asecond oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′portion complementary to a second region of the target nucleic aciddownstream of and contiguous to the first portion. In some embodiments,the 3′ portion of the second oligonucleotide comprises a 3′ terminalnucleotide not complementary to the target nucleic acid. In preferredembodiments, the 3′ portion of the second oligonucleotide consists of asingle nucleotide not complementary to the target nucleic acid. In someembodiments, the kit further comprises a solid support. For example, insome embodiments, the first and/or second oligonucleotide is attached tosaid solid support. In some embodiments, the kit further comprises abuffer solution. In some preferred embodiments, the buffer solutioncomprises a source of divalent cations (e.g., Mn²⁺ and/or Mg²⁺ ions). Insome specific embodiments, the kit further comprises a thirdoligonucleotide complementary to a third portion of the target nucleicacid upstream of the first portion of the first target nucleic acid. Inyet other embodiments, the kit further comprises a target nucleic acid.In some embodiments, the kit further comprises a second target nucleicacid. In yet other embodiments, the kit further comprises a thirdoligonucleotide comprising a 5′ portion complementary to a first regionof the second target nucleic acid. In some specific embodiments, the 3′portion of the third oligonucleotide is covalently linked to the secondtarget nucleic acid. In other specific embodiments, the second targetnucleic acid further comprises a 5′ portion, wherein the 5′ portion ofthe second target nucleic acid is the third oligonucleotide. In stillother embodiments, the kit further comprises an ARRESTOR molecule (e.g.,ARRESTOR oligonucleotide).

[0047] The present invention further provides a composition comprising acleavage structure, the cleavage structure comprising: a) a targetnucleic acid, the target nucleic acid having a first region, a secondregion, a third region and a fourth region, wherein the first region islocated adjacent to and downstream from the second region, the secondregion is located adjacent to and downstream from the third region andthe third region is located adjacent to and downstream from the fourthregion; b) a first oligonucleotide complementary to the fourth region ofthe target nucleic acid; c) a second oligonucleotide having a 5′ portionand a 3′ portion wherein the 5′ portion of the second oligonucleotidecontains a sequence complementary to the second region of the targetnucleic acid and wherein the 3′ portion of the second oligonucleotidecontains a sequence complementary to the third region of the targetnucleic acid; and d) a third oligonucleotide having a 5′ portion and a3′ portion wherein the 5′ portion of the third oligonucleotide containsa sequence complementary to the first region of the target nucleic acidand wherein the 3′ portion of the third oligonucleotide contains asequence complementary to the second region of the target nucleic acid.

[0048] The present invention is not limited by the length of the fourregions of the target nucleic acid. In one embodiment, the first regionof the target nucleic acid has a length of 11 to 50 nucleotides. Inanother embodiment, the second region of the target nucleic acid has alength of one to three nucleotides. In another embodiment, the thirdregion of the target nucleic acid has a length of six to ninenucleotides. In yet another embodiment, the fourth region of the targetnucleic acid has a length of 6 to 50 nucleotides.

[0049] The invention is not limited by the nature or composition of theof the first, second, third and fourth oligonucleotides; theseoligonucleotides may comprise DNA, RNA, PNA and combinations thereof aswell as comprise modified nucleotides, universal bases, adducts, etc.Further, one or more of the first, second, third and the fourtholigonucleotides may contain a dideoxynucleotide at the 3′ terminus.

[0050] In one preferred embodiment, the target nucleic acid is notcompletely complementary to at least one of the first, the second, thethird and the fourth oligonucleotides. In a particularly preferredembodiment, the target nucleic acid is not completely complementary tothe second oligonucleotide.

[0051] As noted above, the present invention contemplates the use ofstructure-specific nucleases in detection methods. In one embodiment,the present invention provides a method of detecting the presence of atarget nucleic acid molecule by detecting non-target cleavage productscomprising: a) providing: i) a cleavage means, ii) a source of targetnucleic acid, the target nucleic acid having a first region, a secondregion, a third region and a fourth region, wherein the first region islocated adjacent to and downstream from the second region, the secondregion is located adjacent to and downstream from the third region andthe third region is located adjacent to and downstream from the fourthregion; iii) a first oligonucleotide complementary to the fourth regionof the target nucleic acid; iv) a second oligonucleotide having a 5′portion and a 3′ portion wherein the 5′ portion of the secondoligonucleotide contains a sequence complementary to the second regionof the target nucleic acid and wherein the 3′ portion of the secondoligonucleotide contains a sequence complementary to the third region ofthe target nucleic acid; iv) a third oligonucleotide having a 5′ and a3′ portion wherein the 5′ portion of the third oligonucleotide containsa sequence complementary to the first region of the target nucleic acidand wherein the 3′ portion of the third oligonucleotide contains asequence complementary to the second region of the target nucleic acid;b) mixing the cleavage means, the target nucleic acid, the firstoligonucleotide, the second oligonucleotide and the thirdoligonucleotide to create a reaction mixture under reaction conditionssuch that the first oligonucleotide is annealed to the fourth region ofthe target nucleic acid and wherein at least the 3′ portion of thesecond oligonucleotide is annealed to the target nucleic acid andwherein at least the 5′ portion of the third oligonucleotide is annealedto the target nucleic acid so as to create a cleavage structure andwherein cleavage of the cleavage structure occurs to generate non-targetcleavage products, each non-target cleavage product having a 3′-hydroxylgroup; and c) detecting the non-target cleavage products.

[0052] The invention is not limited by the nature of the target nucleicacid. In one embodiment, the target nucleic acid comprisessingle-stranded DNA. In another embodiment, the target nucleic acidcomprises double-stranded DNA and prior to step c), the reaction mixtureis treated such that the double-stranded DNA is rendered substantiallysingle-stranded. In another embodiment, the target nucleic acidcomprises RNA and the first and second oligonucleotides comprise DNA.

[0053] The invention is not limited by the nature of the cleavage means.In one embodiment, the cleavage means is a structure-specific nuclease;particularly preferred structure-specific nucleases are thermostablestructure-specific nucleases.

[0054] In another preferred embodiment the thermostable structurespecific nuclease is a chimerical nuclease.

[0055] In an alternative preferred embodiment, the detection of thenon-target cleavage products comprises electrophoretic separation of theproducts of the reaction followed by visualization of the separatednon-target cleavage products.

[0056] In another preferred embodiment, one or more of the first,second, and third oligonucleotides contain a dideoxynucleotide at the 3′terminus. When dideoxynucleotide-containing oligonucleotides areemployed, the detection of the non-target cleavage products preferablycomprises: a) incubating the non-target cleavage products with atemplate-independent polymerase and at least one labeled nucleosidetriphosphate under conditions such that at least one labeled nucleotideis added to the 3′-hydroxyl group of the non-target cleavage products togenerate labeled non-target cleavage products; and b) detecting thepresence of the labeled non-target cleavage products. The invention isnot limited by the nature of the template-independent polymeraseemployed; in one embodiment, the template-independent polymerase isselected from the group consisting of terminal deoxynucleotidyltransferase (TdT) and poly A polymerase. When TdT or polyA polymeraseare employed in the detection step, the second oligonucleotide maycontain a 5′ end label, the 5′ end label being a different label thanthe label present upon the labeled nucleoside triphosphate. Theinvention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3amidite, Cy5 amidite and digoxigenin.

[0057] In another embodiment, detecting the non-target cleavage productscomprises: a) incubating the non-target cleavage products with atemplate-independent polymerase and at least one nucleoside triphosphateunder conditions such that at least one nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generate tailednon-target cleavage products; and b) detecting the presence of thetailed non-target cleavage products. The invention is not limited by thenature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerases are employed in thedetection step, the second oligonucleotide may contain a 5′ end label.The invention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein,REDMOND RED dye, Cy3 amidite, Cy5 amidite and digoxigenin.

[0058] In a preferred embodiment, the reaction conditions compriseproviding a source of divalent cations; particularly preferred divalentcations are Mn²⁺ and Mg²⁺ ions.

[0059] The present invention further provides a method of detecting thepresence of a target nucleic acid molecule by detecting non-targetcleavage products comprising: a) providing: i) a cleavage means, ii) asource of target nucleic acid, the target nucleic acid having a firstregion, a second region and a third region, wherein the first region islocated adjacent to and downstream from the second region and whereinthe second region is located adjacent to and downstream from the thirdregion; iii) a first oligonucleotide having a 5′ and a 3′ portionwherein the 5′ portion of the first oligonucleotide contains a sequencecomplementary to the second region of the target nucleic acid andwherein the 3′ portion of the first oligonucleotide contains a sequencecomplementary to the third region of the target nucleic acid; iv) asecond oligonucleotide having a length between eleven to fifteennucleotides and further having a 5′ and a 3′ portion wherein the 5′portion of the second oligonucleotide contains a sequence complementaryto the first region of the target nucleic acid and wherein the 3′portion of the second oligonucleotide contains a sequence complementaryto the second region of the target nucleic acid; b) mixing the cleavagemeans, the target nucleic acid, the first oligonucleotide and the secondoligonucleotide to create a reaction mixture under reaction conditionssuch that at least the 3′ portion of the first oligonucleotide isannealed to the target nucleic acid and wherein at least the 5′ portionof the second oligonucleotide is annealed to the target nucleic acid soas to create a cleavage structure and wherein cleavage of the cleavagestructure occurs to generate non-target cleavage products, eachnon-target cleavage product having a 3′-hydroxyl group; and c) detectingthe non-target cleavage products. In a preferred embodiment the cleavagemeans is a structure-specific nuclease, preferably a thermostablestructure-specific nuclease.

[0060] The invention is not limited by the length of the various regionsof the target nucleic acid. In a preferred embodiment, the second regionof the target nucleic acid has a length between one to five nucleotides.In another preferred embodiment, one or more of the first and the secondoligonucleotides contain a dideoxynucleotide at the 3′ terminus. Whendideoxynucleotide-containing oligonucleotides are employed, thedetection of the non-target cleavage products preferably comprises: a)incubating the non-target cleavage products with a template-independentpolymerase and at least one labeled nucleoside triphosphate underconditions such that at least one labeled nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generatelabeled non-target cleavage products; and b) detecting the presence ofthe labeled non-target cleavage products. The invention is not limitedby the nature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase is employed in the detectionstep, the second oligonucleotide may contain a 5′ end label, the 5′ endlabel being a different label than the label present upon the labelednucleoside triphosphate. The invention is not limited by the nature ofthe 5′ end label; a wide variety of suitable 5′ end labels are known tothe art and include biotin, fluorescein, tetrachlorofluorescein,hexachlorofluorescein, REDMOND RED dye, Cy3 amidite, Cy5 amidite anddigoxigenin.

[0061] In another embodiment, detecting the non-target cleavage productscomprises: a) incubating the non-target cleavage products with atemplate-independent polymerase and at least one nucleoside triphosphateunder conditions such that at least one nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generate tailednon-target cleavage products; and b) detecting the presence of thetailed non-target cleavage products. The invention is not limited by thenature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerases are employed in thedetection step, the second oligonucleotide may contain a 5′ end label.The invention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein,REDMOND RED dye, Cy3 amidite, Cy5 amidite and digoxigenin.

[0062] The novel detection methods of the invention may be employed forthe detection of target DNAs and RNAs including, but not limited to,target DNAs and RNAs comprising wild type and mutant alleles of genes,including genes from humans or other animals that are or may beassociated with disease or cancer. In addition, the methods of theinvention may be used for the detection of and/or identification ofstrains of microorganisms, including bacteria, fungi, protozoa, ciliatesand viruses (and in particular for the detection and identification ofRNA viruses, such as HCV).

[0063] The present invention further provides novel enzymes designed fordirect detection, characterization and quantitation of nucleic acids,particularly RNA. The present invention provides enzymes that recognizespecific nucleic acid cleavage structures formed on a target RNAsequence and that cleave the nucleic acid cleavage structure in asite-specific manner to produce non-target cleavage products. Thepresent invention provides enzymes having an improved ability tospecifically cleave a DNA member of a complex comprising DNA and RNAnucleic acid strands.

[0064] For example, the present invention provides DNA polymerases thatare altered in structure relative to the native DNA polymerases, suchthat they exhibit altered (e.g., improved) performance in detectionassays based on the cleavage of a structure comprising nucleic acid(e.g., RNA). In particular, the altered polymerases of the presentinvention exhibit improved performance in detection assays based on thecleavage of a DNA member of a cleavage structure (e.g., an invasivecleavage structure) that comprises an RNA target strand.

[0065] The improved performance in a detection assay may arise from anyone of, or a combination of several improved features. For example, inone embodiment, the enzyme of the present invention may have an improvedrate of cleavage (k_(cat)) on a specific targeted structure, such that alarger amount of a cleavage product may be produced in a given timespan. In another embodiment, the enzyme of the present invention mayhave a reduced activity or rate in the cleavage of inappropriate ornon-specific structures. For example, in certain embodiments of thepresent invention, one aspect of improvement is that the differentialbetween the detectable amount of cleavage of a specific structure andthe detectable amount of cleavage of any alternative structures isincreased. As such, it is within the scope of the present invention toprovide an enzyme having a reduced rate of cleavage of a specific targetstructure compared to the rate of the native enzyme, and having afurther reduced rate of cleavage of any alternative structures, suchthat the differential between the detectable amount of cleavage of thespecific structure and the detectable amount of cleavage of anyalternative structures is increased. However, the present invention isnot limited to enzymes that have an improved differential.

[0066] In a preferred embodiment, the enzyme of the present invention isa DNA polymerase having an altered nuclease activity as described above,and also having altered synthetic activity, compared to that of anynative DNA polymerase from which the enzyme has been derived. It isespecially preferred that the DNA polymerase is altered such that itexhibits reduced synthetic activity as well as improved nucleaseactivity on RNA targets, compared to that of the native DNA polymerase.Enzymes and genes encoding enzymes having reduced synthetic activityhave been described (See e.g., Kaiser et al., J. Biol. Chem., 274:21387[1999], Lyamichev et al., Prot. Natl. Acad. Sci., 96:6143 [1999], U.S.Pat. Nos. 5,541,311, 5,614,402, 5,795,763 and 6,090,606, incorporatedherein by reference in their entireties). The present inventioncontemplates combined modifications, such that the resulting 5′nucleases are without interfering synthetic activity, and have improvedperformance in RNA detection assays.

[0067] The present invention contemplates a DNA sequence encoding a DNApolymerase altered in sequence relative to the native sequence, suchthat it exhibits altered nuclease activity from that of the native DNApolymerase. For example, in one embodiment, the DNA sequence encodes anenzyme having an improved rate of cleavage (k_(cat)) on a specifictargeted structure, such that a larger amount of a cleavage product maybe produced in a given time span. In another embodiment, the DNA encodesan enzyme having a reduced activity or rate in the cleavage ofinappropriate or non-specific structures. In certain embodiments, oneaspect of improvement is that the differential between the detectableamount of cleavage of a specific structure and the detectable amount ofcleavage of any alternative structures is increased. It is within thescope of the present invention to provide a DNA encoding an enzymehaving a reduced rate of cleavage of a specific target structurecompared to the rate of the native enzyme, and having a further reducedrate of cleavage of any alternative structures, such that thedifferential between the detectable amount of cleavage of the specificstructure and the detectable amount of cleavage of any alternativestructures is increased. However, the present invention is not limitedto polymerases that have an improved differential.

[0068] In a preferred embodiment, the DNA sequence encodes a DNApolymerase having the altered nuclease activity described above, andalso having altered synthetic activity, compared to that of any nativeDNA polymerase from which the improved enzyme is derived. It isespecially preferred that the encoded DNA polymerase is altered suchthat it exhibits reduced synthetic activity as well as improved nucleaseactivity oil RNA targets, compared to that of the native DNA polymerase.

[0069] It is not intended that the invention be limited by the nature ofthe alteration required to introduce altered nuclease activity. Nor isit intended that the invention be limited by the extent of either thealteration, or in the improvement observed. If the polymerase is alsoaltered so as to be synthesis modified, it is not intended that theinvention be limited by the polymerase activity of the modified orunmodified protein, or by the nature of the alteration to render thepolymerase synthesis modified.

[0070] The present invention contemplates structure-specific nucleasesfrom a variety of sources, including, but not limited to, mesophilic,psychrophilic, thermophilic, and hyperthermophilic organisms. Thepreferred structure-specific nucleases are thermostable. Thermostablestructure-specific nucleases are contemplated as particularly useful inthat they allow the INVADER assay (See e.g., U.S. Pat. Nos. 5,846,717,5,985,557, 5,994,069, 6,001,567, and 6,090,543 and PCT Publications WO97/27214 and WO 98/42873, incorporated herein by reference in theirentireties) to be operated near the melting temperature (T_(m)) of thedownstream probe oligonucleotide, so that cleaved and uncleaved probesmay cycle on and off the target during the course of the reaction. Inone embodiment, the thermostable structure-specific enzymes arethermostable 5′ nucleases that are selected from the group comprisingaltered polymerases derived from the native polymerases of Thermusspecies, including, but not limited to, Thermus aquaticus, Thermusflavus, Thermus thermophilus, Thermus filiformus, and Thermusscotoductus. However, the invention is not limited to the use ofthermostable 5′ nucleases. For example, certain embodiments of thepresent invention utilize short oligonucleotide probes that may cycle onand off of the target at low temperatures, allowing the use ofnon-thermostable enzymes.

[0071] In some preferred embodiments, the present invention provides acomposition comprising an enzyme, wherein the enzyme comprises aheterologous functional domain, wherein the heterologous functionaldomain provides altered (e.g., improved) functionality in a nucleic acidcleavage assay. The present invention is not limited by the nature ofthe nucleic acid cleavage assay. For example, nucleic acid cleavageassays include any assay in which a nucleic acid is cleaved, directly orindirectly, in the presence of the enzyme. In certain preferredembodiments, the nucleic acid cleavage assay is an invasive cleavageassay. In particularly preferred embodiments, the cleavage assayutilizes a cleavage structure having at least one RNA component. Inanother particularly preferred embodiment, the cleavage assay utilizes acleavage structure having at least one RNA component, wherein a DNAmember of the cleavage structure is cleaved.

[0072] In some preferred embodiments, the enzyme comprises a 5′ nucleaseor a polymerase. In certain preferred embodiments, the 5′ nucleasecomprises a thermostable 5′ nuclease. In other preferred embodiments,the polymerase is altered in sequence relative to a naturally occurringsequence of a polymerase such that it exhibits reduced DNA syntheticactivity from that of the naturally occurring poly erase. In certainpreferred embodiments, the polymerase comprises a thermostablepolymerase (e.g., a polymerase from a Thermus species including, but notlimited to, Thermus aquaticus, Thermus flavus, Thermus thermophilus,Thermus filiformus, and Thermus scotoductus).

[0073] The present invention is not limited by the nature of the alteredfunctionality provided by the heterologous functional domain.Illustrative examples of alterations include, but are not limited to,enzymes where the heterologous functional domain comprises an amino acidsequence (e.g., one or more amino acids) that provides an improvednuclease activity, an improved substrate binding activity and/orimproved background specificity in a nucleic acid cleavage assay.

[0074] The present invention is not limited by the nature of theheterologous functional domain. For example, in some embodiments, theheterologous functional domain comprises two or more amino acids from apolymerase domain of a polymerase (e.g., introduced into the enzyme byinsertion of a chimeric functional domain or created by mutation). Incertain preferred embodiment, at least one of the two or more aminoacids is from a palm or thumb region of the polymerase domain. Thepresent invention is not limited by the identity of the polymerase fromwhich the two or more amino acids are selected. In certain preferredembodiments, the polymerase comprises Thermus thermophilus polymerase.In particularly preferred embodiments, the two or more amino acids arefrom amino acids 300-650 of SEQ ID NO:1.

[0075] The novel enzymes of the invention may be employed for thedetection of target DNAs and RNAs including, but not limited to, targetDNAs and RNAs comprising wild type and mutant alleles of genes,including, but not limited to, genes from humans, other animal, orplants that are or may be associated with disease or other conditions.In addition, the enzymes of the invention may be used for the detectionof and/or identification of strains of microorganisms, includingbacteria, fungi, protozoa, ciliates and viruses (and in particular forthe detection and identification of viruses having RNA genomes, such asthe Hepatitis C and Human Immunodeficiency viruses). For example, thepresent invention provides methods for cleaving a nucleic acidcomprising providing: an enzyme of the present invention and a substratenucleic acid; and exposing the substrate nucleic acid to the enzyme(e.g., to produce a cleavage product that may be detected).

[0076] In one embodiment, the present invention provides a thermostable5′ nuclease having an amino acid sequence selected from the groupcomprising SEQ ID NOS:2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 341,346, 348, 351, 353, 359, 365, 367, 369, 374, 376, 380, 384, 388, 392,396, 400, 402, 406, 408, 410, 412, 416, 418, 420, 424, 427, 429, 432,436, 440, 444, 446, 448, 450, 456, 460, 464, 468, 472, 476, 482, 485,488, 491, 494, 496, 498, 500, 502, 506, 510, 514, 518, 522, 526, 530,534, 538, 542, 544, 550, 553, 560, 564, 566, 568, 572, 574, 576, 578,580, 582, 584, 586, 588, 590, 2641-2674, 2710, 2712, 2714, 2717, 2719,2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744,2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2766, 2768, 2770,2772, 2774, 2776, 2778, 2780, 2781, 2782, 2783, 2785, 2787, 2789, 2791,2793, 2795, 2797, 2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815,2817, 2819, 2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835, and 2837. Inanother embodiment, the 5′ nuclease is encoded by a DNA sequenceselected from the group comprising of SEQ ID NOS:69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 340,345, 347, 350, 352, 358, 364, 366, 368, 373, 375, 379, 383, 387, 391,395, 399, 401, 405, 407, 409, 411, 415, 417, 419, 423, 426, 428, 431,435, 439, 443, 445, 447, 449, 452, 454, 455, 459, 463, 467, 471, 475,481, 484, 495, 497, 499, 501, 505, 509, 513, 517, 521, 525, 529, 533,537, 541, 543, 549, 552, 559, 563, 565, 567, 571, 573, 575, 577, 579,581, 583, 585, 587, 589, 2675-2709, 2711, 2713, 2715, 2716, 2718, 2720,2721, 2723, 2725, 2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 27432745, 2747, 2749, 2751, 2753, 2755, 2757, 2759, 2761, 2763, 2764, 2765,2767, 2769, 2771, 2773, 2775, 2777, 2779, 2784, 2786, 2788, 2790, 2792,2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816,2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834, and 2836.

[0077] The present invention also provides a recombinant DNA vectorcomprising DNA having a nucleotide sequence encoding a 5′ nuclease, thenucleotide sequence selected from the group comprising SEQ ID NOS: 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 340, 345, 347, 350, 352, 358, 364, 366, 368, 373, 375,379, 383, 387, 391, 395, 399, 401, 405, 407, 409, 411, 415, 417, 419,423, 426, 428, 431, 435, 439, 443, 445, 447, 449, 452, 454, 455, 459,463, 467, 471, 475, 481, 484, 495, 497, 499, 501, 505, 509, 513, 517,521, 525, 529, 533, 537, 541, 543, 549, 552, 559, 563, 565, 567, 571,573, 575, 577, 579, 581, 583, 585, 587, 589, 2675-2709, 2711, 2713,2715, 2716, 2718, 2720, 2721, 2723, 2725, 2727, 2729, 2731, 2733, 2735,2737, 2739, 2741, 2743 2745, 2747, 2749, 2751, 2753, 2755, 2757, 2759,2761, 2763, 2764, 2765, 2767, 2769, 2771, 2773, 2775, 2777, 2779, 2784,2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808,2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832,2834, and 2836. In a preferred embodiment, the invention provides a hostcell transformed with a recombinant DNA vector comprising DNA having anucleotide sequence encoding a structure-specific nuclease, thenucleotide selected from the group comprising sequence SEQ ID NOS: 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 340, 345, 347, 350, 352, 358, 364, 366, 368, 373, 375,379, 383, 387, 391, 395, 399, 401, 405, 407, 409, 411, 415, 417, 419,423, 426, 428, 431, 435, 439, 443, 445, 447, 449, 452, 454, 455, 459,463, 467, 471, 475, 481, 484, 495, 497, 499, 501, 505, 509, 513, 517,521, 525, 529, 533, 537, 541, 543, 549, 552, 559, 563, 565, 567, 571,573, 575, 577, 579, 581, 583, 585, 587, 589, 2675-2709, 2711, 2713,2715, 2716, 2718, 2720, 2721, 2723, 2725, 2727, 2729, 2731, 2733, 2735,2737, 2739, 2741, 2743 2745, 2747, 2749, 2751, 2753, 2755, 2757, 2759,2761, 2763, 2764, 2765, 2767, 2769, 2771, 2773, 2775, 2777, 2779, 2784,2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808,2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832,2834, and 2836. The invention is not limited by the nature of the hostcell employed. The art is well aware of expression vectors suitable forthe expression of nucleotide sequences encoding structure-specificnucleases that can be expressed in a variety of prokaryotic andeukaryotic host cells. In a preferred embodiment, the host cell is anEscherichia coli cell.

[0078] The present invention provides a method of altering S nucleaseenzymes relative to native 5′ nuclease enzymes, such that they exhibitimproved performance in detection assays based on the cleavage of astructure comprising RNA. In particular, the altered 5′ nucleasesproduced by the method of the present invention exhibit improvedperformance in detection assays based on the cleavage of a DNA member ofa cleavage structure (e.g., an invasive cleavage structure) thatcomprises an RNA target strand. The improved 5′ nucleases resulting fromthe methods of the present invention may be improved in any of the waysdiscussed herein. Examples of processes for assessing improvement in anycandidate enzyme are provided.

[0079] For example, the present invention provides methods for producingan altered enzyme with improved functionality in a nucleic acid cleavageassay comprising: providing an enzyme and a nucleic acid test substrate;introducing a heterologous functional domain into the enzyme to producean altered enzyme; contacting the altered enzyme with the nucleic acidtest substrate to produce cleavage products; and detecting the cleavageproducts. In some embodiments, the introduction of the heterologousfunctional domain comprises mutating one or more amino acids of theenzyme. In other embodiments, the introduction of the heterologousfunctional domain into the enzyme comprises adding a functional domainfrom a protein (e.g., another enzyme) into the enzyme (e.g. substitutingfunctional domains by removing a portion of the enzyme sequence prior toadding the functional domain of the protein). In preferred embodiments,the nucleic acid test substrate comprises a cleavage structure. Inparticularly preferred embodiment, the cleavage structure comprises anRNA target nucleic acid. In yet other preferred embodiments, thecleavage structure comprises an invasive cleavage structure.

[0080] The present invention also provides nucleic acid treatment kits.One preferred embodiment is a kit comprising a composition comprising atleast one improved 5′ nuclease. Another preferred embodiment provides akit comprising: a) a composition comprising at least one improved 5′nuclease; and b) an INVADER oligonucleotide and a signal probeoligonucleotide. In some embodiments of the kits of the presentinvention, the improved 5′ nuclease is derived from a DNA polymerasefrom a eubacterial species. In further embodiments, the eubacterialspecies is a thermophile. In still further embodiments, the thermophileis of the genus Thermus. In still further embodiments, the thermophileis selected from the group consisting of Thermus aquaticus, Thermusflavus, Thermus thermophilus, Thermus filiformus, and Thermusscotoductus. In preferred embodiments, the improved 5′ nuclease isencoded by DNA selected from the group comprising SEQ ID NOS: 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 340, 345, 347, 350, 352, 358, 364, 366, 368, 373, 375,379, 383, 387, 391, 395, 399, 401, 405, 407, 409, 411, 415, 417, 419,423, 426, 428, 431, 435, 439, 443, 445, 447, 449, 452, 454, 455, 459,463, 467, 471, 475, 481, 484, 495, 497, 499, 501, 505, 509, 513, 517,521, 525, 529, 533, 537, 541, 543, 549, 552, 559, 563, 565, 567, 571,573, 575, 577, 579, 581, 583, 585, 587, 589, 2675-2709, 2711, 2713,2715, 2716, 2718, 2720, 2721, 2723, 2725, 2727, 2729, 2731, 2733, 2735,2737, 2739, 2741, 2743 2745, 2747, 2749, 2751, 2753, 2755, 2757, 2759,2761, 2763, 2764, 2765, 2767, 2769, 2771, 2773, 2775, 2777, 2779, 2784,2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808,2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832,2834, and 2836. In yet other preferred embodiments, the kits furthercomprise reagents for detecting a nucleic acid cleavage product. Infurther preferred embodiments, the reagents for detecting a cleavageproduct comprise oligonucleotides for use in a subsequent invasivecleavage reaction (See e.g., U.S. Pat. No. 5,994,069). In particularlypreferred embodiments, the reagents for the subsequent invasive cleavagereaction comprise a probe labeled with moieties that produce afluorescence resonance energy transfer (FRET) effect.

[0081] The present invention also provides methods for treating nucleicacid, comprising: a) providing: a first structure-specific nucleaseconsisting of an endonuclease in a solution containing manganese; and anucleic acid substrate; b) treating the nucleic acid substrate withincreased temperature such that the substrate is substantiallysingle-stranded; c) reducing the temperature under conditions such thatthe single-stranded substrate forms one or more cleavage structures; d)reacting the cleavage means with the cleavage structures so that one ormore cleavage products are produced; and e) detecting the one or morecleavage products. In some embodiments of the methods, the endonucleaseincludes, but is not limited to, CLEAVASE BN enzyme, Thermus aquaticusDNA polymerase, Thermus thermophilus DNA polymerase, Escherichia coliExo III, and the Saccharomyces cerevisiae Rad1/Rad10 complex. In yetother preferred embodiments, the nuclease is a 5′ nuclease derived froma thermostable DNA polymerase altered in amino acid sequence such thatit exhibits reduced DNA synthetic activity from that of the wild-typeDNA polymerase but retains substantially the same 5′ nuclease activityof the wild-type DNA polymerase. In yet other embodiments, the nucleicacid is selected from the group consisting of RNA and DNA. In furtherembodiments, the nucleic acid of step (a) is double stranded.

[0082] The present invention also provides nucleic acid treatment kits,comprising: a) a composition comprising at least one purified FEN-1endonuclease; and b) a solution containing manganese. In someembodiments of the kits, the purified FEN-1 endonuclease is selectedfrom the group consisting Pyrococcus woesei FEN-1 endonuclease,Pyrococcus furiosus FEN-1 endonuclease, Methanococcus jannaschii FEN-1endonuclease, Methanobacterium thermoautotrophicum FEN-1 endonuclease,Archaeoglobus fulgidus FEN-1, Sulfolobus solfataricus, Pyrobaculumaerophilum, Thermococcus litoralis, Archaeaglobus veneficus,Archaeaglobus profundus, Acidianus brierlyi, Acidianus ambivalens,Desulfurococcus amylolyticus, Desulfurococcus mobilis, Pyrodictiumbrockii, Thermococcus gorgonarius, Thermococcus zilligii, Methanopyruskandleri, Methanococcus igneus, Pyrococcus horikoshii, Aeropyrum pernix,and chimerical FEN-1 endonucleases. In other embodiments, the kitsfurther comprise at least one second structure-specific nuclease. Insome preferred embodiments, the second nuclease is a 5′ nuclease derivedfrom a thermostable DNA polymerase altered in amino acid sequence suchthat it exhibits reduced DNA synthetic activity from that of thewild-type DNA polymerase but retains substantially the same 5′ nucleaseactivity of the wild-type DNA polymerase. In yet other embodiments ofthe kits, the portion of the amino acid sequence of the second nucleaseis homologous to a portion of the amino acid sequence of a thermostableDNA polymerase derived from a eubacterial thermophile of the genusThermus. In further embodiments, the thermophile is selected from thegroup consisting of Thermus aquaticus, Thermus flavus and Thermusthermophilus. In yet other preferred embodiments, the kits furthercomprise reagents for detecting the cleavage products.

[0083] The present invention further provides any of the compositions,mixtures, methods, and kits described herein, used in conjunction withendonucleases comprising Sulfolobus solfataricus, Pyrobaculumaerophilum, Thermococcus litoralis, Archaeaglobus veneficus,Archaeaglobus profundus, Acidianus brierlyi, Acidianus ambivalens,Desulfurococcus amylolyticus, Desulfurococcus mobilis, Pyrodictiumbrockii, Thermococcus gorgonarius, Thermococcus zilligii, Methanopyruskandleri, Methanococcus igneus, Pyrococcus horikoshii, and Aeropyrumpernix endonucleases. These include compositions comprising purifiedFEN-1 endonucleases from the organisms (including specific endonucleasesdescribed by sequences provided herein, as well as, variants andhomologues), kits comprising these compositions, composition comprisingchimerical endonucleases comprising at least a portion of theendonucleases from these organisms, kits comprising such compositions,compositions comprising nucleic acids encoding the endonucleases fromthese organisms (including vectors and host cells), kits comprising suchcompositions, antibodies generated to the endonucleases, mixturescomprising endonucleases from these organisms, methods of using theendonuclease in cleavage assays (e.g., invasive cleavage assays, CFLP,etc.), and kits containing components useful for such methods. Examplesdescribing the generation, structure, use, and characterization of theseendonucleases are provided herein.

[0084] The present invention also provides methods for improving themethods and enzymes disclosed herein. For example, the present inventionprovides methods of improving enzymes for any intended purpose (e.g.,use in cleavage reactions, amplification reactions, binding reactions,or any other use) comprising the step of providing an enzyme disclosedherein and modifying the enzyme (e.g., altering the amino acid sequence,adding or subtracting sequence, adding post-translational modifications,adding any other component whether biological or not, or any othermodification). Likewise, the present invention provides methods forimproving the methods disclosed herein comprising, conducting the methodsteps with one or more changes (e.g., change in a composition providedin the method, change in the order of the steps, or addition orsubtraction of steps).

[0085] The improved performance in a detection assay may arise from anyone of, or a combination of several improved features. For example, inone embodiment, the enzyme of the present invention may have an improvedrate of cleavage (k_(cat)) on a specific targeted structure, such that alarger amount of a cleavage product may be produced in a given timespan. In another embodiment, the enzyme of the present invention mayhave a reduced activity or rate in the cleavage of inappropriate ornon-specific structures. For example, in certain embodiments of thepresent invention, one aspect of improvement is that the differentialbetween the detectable amount of cleavage of a specific structure andthe detectable amount of cleavage of any alternative structures isincreased. As such, it is within the scope of the present invention toprovide an enzyme having a reduced rate of cleavage of a specific targetstructure compared to the rate of the native enzyme, and having afurther reduced rate of cleavage of any alternative structures, suchthat the differential between the detectable amount of cleavage of thespecific structure and the detectable amount of cleavage of anyalternative structures is increased. However, the present invention isnot limited to enzymes that have an improved differential.

[0086] In some preferred embodiments, the present invention provides acomposition comprising an enzyme, wherein the enzyme comprises aheterologous functional domain, wherein the heterologous functionaldomain provides altered (e.g., improved) functionality in a nucleic acidcleavage assay. The present invention is not limited by the nature ofthe nucleic acid cleavage assay. For example, nucleic acid cleavageassays include any assay in which a nucleic acid is cleaved, directly orindirectly, in the presence of the enzyme. In certain preferredembodiments, the nucleic acid cleavage assay is an invasive cleavageassay. In particularly preferred embodiments, the cleavage assayutilizes a cleavage structure having at least one RNA component. Inanother particularly preferred embodiment, the cleavage assay utilizes acleavage structure having at least one RNA component, wherein a DNAmember of the cleavage structure is cleaved.

[0087] The present invention is not limited by the nature of the alteredfunctionality provided by the heterologous functional domain.Illustrative examples of alterations include, but are not limited to,enzymes where the heterologous functional domain comprises an amino acidsequence (e.g., one or more amino acids) that provides an improvednuclease activity, an improved substrate binding activity and/orimproved background specificity in a nucleic acid cleavage assay.

[0088] The present invention is not limited by the nature of theheterologous functional domain. For example, in some embodiments, theheterologous functional domain comprises two or more amino acids from apolymerase domain of a polymerase (e.g., introduced into the enzyme byinsertion of a chimerical functional domain or created by mutation). Incertain preferred embodiment, at least one of the two or more aminoacids is from a palm or thumb region of the polymerase domain. Thepresent invention is not limited by the identity of the polymerase fromwhich the two or more amino acids are selected. In certain preferredembodiments, the polymerase comprises Thermus thermophilus polymerase.In particularly preferred embodiments, the two or more amino acids arefrom amino acids 300-650 of SEQ ID NO:1.

[0089] The novel enzymes of the invention may be employed for thedetection of target DNAs and RNAs including, but not limited to, targetDNAs and RNAs comprising wild type and mutant alleles of genes,including, but not limited to, genes from humans, other animal, orplants that are or may be associated with disease or other conditions.In addition, the enzymes of the invention may be used for the detectionof and/or identification of strains of microorganisms, includingbacteria, fungi, protozoa, ciliates and viruses (and in particular forthe detection and identification of viruses having RNA genomes, such asthe Hepatitis C and Human Immunodeficiency viruses). For example, thepresent invention provides methods for cleaving a nucleic acidcomprising providing: an enzyme of the present invention and a substratenucleic acid; and exposing the substrate nucleic acid to the enzyme(e.g., to produce a cleavage product that may be detected). In someembodiments, the substrate nucleic is in a cell lysate sample.

[0090] The present invention also provides a method for detecting thepresence of a target nucleic acid comprising: cleaving an invasivecleavage structure, said invasive cleavage structure comprising an RNAtarget nucleic acid; and detecting the cleavage of the invasive cleavagestructure. Such an assay may comprise a multiplex assay, whereinmultiple invasive cleavage structures are cleaved. Such structuresinclude structures formed on different target nucleic acids, as well as,structures formed on different locations of the sample target nucleicacid. In some embodiments, the target nucleic acid comprises a firstregion and a second region, said second region downstream of andcontiguous to said first region. In some embodiments, the invasivecleavage structure comprises the target nucleic acid, a firstoligonucleotide, and a second oligonucleotide, wherein at least aportion of the first oligonucleotide is completely complementary to thefirst portion of the first target nucleic acid, and wherein the secondoligonucleotide comprises a 3′ portion and a 5′ portion, wherein the 5′portion is completely complementary to said second portion of the targetnucleic acid. In some embodiments, the 3′ portion of the secondoligonucleotide comprises a 3′ terminal nucleotide not complementary tosaid target nucleic acid. In some embodiments, the 3′ portion of thesecond oligonucleotide consists of a single nucleotide not complementaryto the target nucleic acid. In some embodiments, the method furthercomprises the steps of forming a second invasive cleavage structurecomprising a non-target cleavage product and cleaving the secondinvasive cleavage structure. In some embodiments, the invasive cleavagestructure or the second invasive cleavage comprises an oligonucleotidecomprising a sequence selected from the group consisting of SEQ IDNO:709-2640. In other embodiments, the invasive cleavage structure orthe second invasive cleavage comprises an oligonucleotide comprising asequence selected from the group consisting of SEQ ID NO:169-211 and619-706. In still other embodiments, the invasive cleavage structure orthe second invasive cleavage comprises an oligonucleotide comprising asequence selected from the group consisting of SEQ ID NO:2837-4004. Insome preferred embodiments, the target nucleic acid comprises acytochrome P450 RNA or a cytokine RNA. In some embodiments, the firstregion or the second region of the target nucleic acid encompasses asplice junction, an exon (or a portion thereof), or an intron (or aportion thereof). In some embodiments, the RNA target nucleic acid isprovided in a cell lysate. In some embodiments, the firstoligonucleotide is covalently attached to the second oligonucleotide.Such oligonucleotides find use, for example, in methods described inU.S. Pat. Nos. 5,714,320 and 5,854,033, herein incorporated by referencein their entireties. The present invention also provides kits containingone or more of the components used in the above methods.

[0091] Definitions

[0092] To facilitate an understanding of the present invention, a numberof terms and phrases are defined below:

[0093] As used herein, the terms “complementary” or “complementarity”are used in reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a target nucleic acid) relatedby the base-pairing rules. For example, for the sequence “5′-A-G-T-3′,”is complementary to the sequence “3′-T-C-A-5′.” Complementarity may be“partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon binding between nucleicacids. Either term may also be used in reference to individualnucleotides, especially within the context of polynucleotides. Forexample, a particular nucleotide within an oligonucleotide may be notedfor its complementarity, or lack thereof, to a nucleotide within anothernucleic acid strand, in contrast or comparison to the complementaritybetween the rest of the oligonucleotide and the nucleic acid strand.Nucleotide analogs used to form non-standard base pairs, whether withanother nucleotide analog (e.g., an IsoC/IsoG base pair), or with anaturally occurring nucleotide (e.g., as described in U.S. Pat. No.5,912,340, herein incorporated by reference in its entirety) are alsoconsidered to be complementary to a base pairing partner within themeaning this definition. Further, when nucleotides are known to formpairs with multiple different bases, e.g., IsoG nucleotide ability topair with IsoC and with T nucleotides, each of the bases with which itcan form a hydrogen-bonded base-pair falls within the meaning of“complementary,” as used herein. “Universal” bases, i.e., those that canform base pairs with several other bases, such as the “wobble” baseinosine, are considered complementary to those bases with which pairscan be formed.

[0094] The term “homology” and “homologous” refers to a degree ofidentity. There may be partial homology or complete homology. Apartially homologous sequence is one that is less than 100% identical toanother sequence.

[0095] As used herein, the term “hybridization” is used in reference tothe pairing of complementary nucleic acids. Hybridization and thestrength of hybridization (i.e., the strength of the association betweenthe nucleic acids) is influenced by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid. “Hybridization” methodsinvolve the annealing of one nucleic acid to another, complementarynucleic acid, i.e., a nucleic acid having a complementary nucleotidesequence. The ability of two polymers of nucleic acid containingcomplementary sequences to find each other and anneal through basepairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modem biology.

[0096] With regard to complementarity, it is important for somediagnostic applications to determine whether the hybridizationrepresents complete or partial complementarity. For example, where it isdesired to detect simply the presence or absence of pathogen DNA (suchas from a virus, bacterium, fungi, mycoplasma, protozoan) it is onlyimportant that the hybridization method ensures hybridization when therelevant sequence is present; conditions can be selected where bothpartially complementary probes and completely complementary probes willhybridize. Other diagnostic applications, however, may require that thehybridization method distinguish between partial and completecomplementarity. It may be of interest to detect genetic polymorphisms.For example, human hemoglobin is composed, in part, of four polypeptidechains. Two of these chains are identical chains of 141 amino acids(alpha chains) and two of these chains are identical chains of 146 aminoacids (beta chains). The gene encoding the beta chain is known toexhibit polymorphism. The normal allele encodes a beta chain havingglutamic acid at the sixth position. The mutant allele encodes a betachain having valine at the sixth position. This difference in aminoacids has a profound (most profound when the individual is homozygousfor the mutant allele) physiological impact known clinically as sicklecell anemia. It is well known that the genetic basis of the amino acidchange involves a single base difference between the normal allele DNAsequence and the mutant allele DNA sequence.

[0097] The complement of a nucleic acid sequence as used herein refersto an oligonucleotide which, when aligned with the nucleic acid sequencesuch that the 5′ end of one sequence is paired with the 3′ end of theother, is in “antiparallel association.” Certain bases not commonlyfound in natural nucleic acids may be included in the nucleic acids ofthe present invention and include, for example, inosine and7-deazaguanine. Complementarity need not be perfect; stable duplexes maycontain mismatched base pairs or unmatched bases. Those skilled in theart of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

[0098] As used herein, the term “T_(m)” is used in reference to the“melting temperature.” The melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf dissociated into single strands. Several equations for calculatingthe T_(m) of nucleic acids are well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (see e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985).Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr.Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry36, 10581-94 (1997) include more sophisticated computations which takestructural and environmental, as well as sequence characteristics intoaccount for the calculation of T_(m).

[0099] As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “weak” or “low”stringency are often required when it is desired that nucleic acids thatare not completely complementary to one another be hybridized orannealed together.

[0100] “High stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42 C in a solution consisting of 5×SSPE (43.8 g/l NaCl,6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH),0.5% SDS, 5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNAfollowed by washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42 Cwhen a probe of about 500 nucleotides in length is employed.

[0101] “Medium stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42 C in a solution consisting of 5×SSPE (43.8 g/l NaCl,6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH),0.5% SDS, 5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNAfollowed by washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42 Cwhen a probe of about 500 nucleotides in length is employed.

[0102] “Low stringency conditions” comprise conditions equivalent tobinding or hybridization at 42 C in a solution consisting of 5×SSPE(43.8 g/l NaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4with NaOH), 0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)]and 100 g/ml denatured salmon sperm DNA followed by washing in asolution comprising 5×SSPE, 0.1% SDS at 42 C when a probe of about 500nucleotides in length is employed.

[0103] The term “gene” refers to a DNA sequence that comprises controland coding sequences necessary for the production of an RNA having anon-coding function (e.g., a ribosomal or transfer RNA), a polypeptideor a precursor. The RNA or polypeptide can be encoded by a full-lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or function is retained.

[0104] The term “wild-type” refers to a gene or a gene product that hasthe characteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified,” “mutant,” or “polymorphic” refers to a gene or gene productthat displays modifications in sequence and or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product. It is noted that naturally-occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

[0105] The term “recombinant DNA vector” as used herein refers to DNAsequences containing a desired heterologous sequence. For example,although the term is not limited to the use of expressed sequences orsequences that encode an expression product, in some embodiments, theheterologous sequence is a coding sequence and appropriate DNA sequencesnecessary for either the replication of the coding sequence in a hostorganism, or the expression of the operably linked coding sequence in aparticular host organism. DNA sequences necessary for expression inprokaryotes include a promoter, optionally an operator sequence, aribosome-binding site and possibly other sequences. Eukaryotic cells areknown to utilize promoters, polyadenlyation signals and enhancers.

[0106] The term “LTR” as used herein refers to the long terminal repeatfound at each end of a provirus (i.e., the integrated form of aretrovirus). The LTR contains numerous regulatory signals includingtranscriptional control elements, polyadenylation signals and sequencesneeded for replication and integration of the viral genome. The viralLTR is divided into three regions called U3, R and U5.

[0107] The U3 region contains the enhancer and promoter elements. The U5region contains the polyadenylation signals. The R (repeat) regionseparates the U3 and U5 regions and transcribed sequences of the Rregion appear at both the 5′ and 3′ ends of the viral RNA.

[0108] The term “oligonucleotide” as used herein is defined as amolecule comprising two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 10-15nucleotides and more preferably at least about 15 to 30 nucleotides. Theexact size will depend on many factors, which in turn depend on theultimate function or use of the oligonucleotide. The oligonucleotide maybe generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, PCR, or a combination thereof.

[0109] Because mononucleotides are reacted to make oligonucleotides in amanner such that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

[0110] When two different, non-overlapping oligonucleotides anneal todifferent regions of the same linear complementary nucleic acidsequence, and the 3′ end of one oligonucleotide points towards the 5′end of the other, the former may be called the “upstream”oligonucleotide and the latter the “downstream” oligonucleotide.Similarly, when two overlapping oligonucleotides are hybridized to thesame linear complementary nucleic acid sequence, with the firstoligonucleotide positioned such that its 5′ end is upstream of the 5′end of the second oligonucleotide, and the 3′ end of the firstoligonucleotide is upstream of the 3′ end of the second oligonucleotide,the first oligonucleotide may be called the “upstream” oligonucleotideand the second oligonucleotide may be called the “downstream”oligonucleotide.

[0111] The term “primer” refers to an oligonucleotide that is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

[0112] A primer is selected to be “substantially” complementary to astrand of specific sequence of the template. A primer must besufficiently complementary to hybridize with a template strand forprimer elongation to occur. A primer sequence need not reflect the exactsequence of the template. For example, a non-complementary nucleotidefragment may be attached to the 5′ end of the primer, with the remainderof the primer sequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

[0113] The term “label” as used herein refers to any atom or moleculethat can be used to provide a detectable (preferably quantifiable)effect, and that can be attached to a nucleic acid or protein. Labelsinclude but are not limited to dyes; radiolabels such as ³²P; bindingmoieties such as biotin; haptens such as digoxgenin; luminogenic,phosphorescent or fluorogenic moieties; and fluorescent dyes alone or incombination with moieties that can suppress or shift emission spectra byfluorescence resonance energy transfer (FRET). Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like. A label may be a charged moiety (positive ornegative charge) or alternatively, may be charge neutral. Labels caninclude or consist of nucleic acid or protein sequence, so long as thesequence comprising the label is detectable.

[0114] The term “signal” as used herein refers to any detectable effect,such as would be caused or provided by a label or an assay reaction.

[0115] As used herein, the term “detector” refers to a system orcomponent of a system, e.g., an instrument (e.g. a camera, fluorimeter,charge-coupled device, scintillation counter, etc.) or a reactive medium(X-ray or camera film, pH indicator, etc.), that can convey to a user orto another component of a system (e.g., a computer or controller) thepresence of a signal or effect. A detector can be a photometric orspectrophotometric system, which can detect ultraviolet, visible orinfrared light, including fluorescence or chemiluminescence; a radiationdetection system; a spectroscopic system such as nuclear magneticresonance spectroscopy, mass spectrometry or surface enhanced Ramanspectrometry; a system such as gel or capillary electrophoresis or gelexclusion chromatography; or other detection systems known in the art,or combinations thereof.

[0116] The term “cleavage structure” as used herein, refers to astructure that is formed by the interaction of at least one probeoligonucleotide and a target nucleic acid, forming a structurecomprising a duplex, the resulting structure being cleavable by acleavage agent, including but not limited to an enzyme. The cleavagestructure is a substrate for specific cleavage by the cleavage means incontrast to a nucleic acid molecule that is a substrate for non-specificcleavage by agents such as phosphodiesterases that cleave nucleic acidmolecules without regard to secondary structure (i.e., no formation of aduplexed structure is required).

[0117] The term “folded cleavage structure” as used herein, refers to aregion of a single-stranded nucleic acid substrate containing secondarystructure, the region being cleavable by an enzymatic cleavage means.The cleavage structure is a substrate for specific cleavage by thecleavage means in contrast to a nucleic acid molecule that is asubstrate for non-specific cleavage by agents such as phosphodiesterasesthat cleave nucleic acid molecules without regard to secondary structure(i.e., no folding of the substrate is required).

[0118] As used herein, the term “folded target” refers to a nucleic acidstrand that contains at least one region of secondary structure (i.e.,at least one double stranded region and at least one single-strandedregion within a single strand of the nucleic acid). A folded target maycomprise regions of tertiary structure in addition to regions ofsecondary structure.

[0119] The term “cleavage means” or “cleavage agent” as used hereinrefers to any means that is capable of cleaving a cleavage structure,including but not limited to enzymes. The cleavage means may includenative DNAPs having 5′ nuclease activity (e.g., Taq DNA polymerase, E.coli DNA polymerase I) and, more specifically, modified DNAPs having 5′nuclease but lacking synthetic activity. “Structure-specific nucleases”or “structure-specific enzymes” are enzymes that recognize specificsecondary structures in a nucleic acid molecule and cleave thesestructures. The cleavage means of the invention cleave a nucleic acidmolecule in response to the formation of cleavage structures; it is notnecessary that the cleavage means cleave the cleavage structure at anyparticular location within the cleavage structure.

[0120] The cleavage means is not restricted to enzymes having solely 5′nuclease activity. The cleavage means may include nuclease activityprovided from a variety of sources including the CLEAVASE enzymes, theFEN-1 endonucleases (including RAD2 and XPG proteins), Taq DNApolymerase and E. coli DNA polymerase I.

[0121] The term “thermostable” when used in reference to an enzyme, suchas a 5′ nuclease, indicates that the enzyme is functional or active(i.e., can perform catalysis) at an elevated temperature, i.e., at about55° C. or higher.

[0122] The term “cleavage products” as used herein, refers to productsgenerated by the reaction of a cleavage means with a cleavage structure(i.e., the treatment of a cleavage structure with a cleavage means).

[0123] The term “target nucleic acid” refers to a nucleic acid moleculecontaining a sequence that has at least partial complementarity with atleast a probe oligonucleotide and may also have at least partialcomplementarity with an INVADER oligonucleotide. The target nucleic acidmay comprise single- or double-stranded DNA or RNA, and may comprisenucleotide analogs, labels, and other modifications.

[0124] The term “probe oligonucleotide” refers to an oligonucleotidethat interacts with a target nucleic acid to form a cleavage structurein the presence or absence of an INVADER oligonucleotide. When annealedto the target nucleic acid, the probe oligonucleotide and target form acleavage structure and cleavage occurs within the probe oligonucleotide.

[0125] The term “non-target cleavage product” refers to a product of acleavage reaction that is not derived from the target nucleic acid. Asdiscussed above, in the methods of the present invention, cleavage ofthe cleavage structure generally occurs within the probeoligonucleotide. The fragments of the probe oligonucleotide generated bythis target nucleic acid-dependent cleavage are “non-target cleavageproducts.”

[0126] The term “INVADER oligonucleotide” refers to an oligonucleotidethat hybridizes to a target nucleic acid at a location near the regionof hybridization between a probe and the target nucleic acid, whereinthe INVADER oligonucleotide comprises a portion (e.g., a chemicalmoiety, or nucleotide-whether complementary to that target or not) thatoverlaps with the region of hybridization between the probe and target.In some embodiments, the INVADER oligonucleotide contains sequences atits 3′ end that are substantially the same as sequences located at the5′ end of a probe oligonucleotide.

[0127] The term “substantially single-stranded” when used in referenceto a nucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

[0128] The term “sequence variation” as used herein refers todifferences in nucleic acid sequence between two nucleic acids. Forexample, a wild-type structural gene and a mutant form of this wild-typestructural gene may vary in sequence by the presence of single basesubstitutions and/or deletions or insertions of one or more nucleotides.These two forms of the structural gene are said to vary in sequence fromone another. A second mutant form of the structural gene may exist. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene.

[0129] The term “liberating” as used herein refers to the release of anucleic acid fragment from a larger nucleic acid fragment, such as anoligonucleotide, by the action of, for example, a 5′ nuclease such thatthe released fragment is no longer covalently attached to the remainderof the oligonucleotide.

[0130] The term “K_(m)” as used herein refers to the Michaelis-Mentenconstant for an enzyme and is defined as the concentration of thespecific substrate at which a given enzyme yields one-half its maximumvelocity in an enzyme catalyzed reaction.

[0131] The term “nucleotide” as used herein includes, but is not limitedto, naturally occurring and/or synthetic nucleotides, nucleotideanalogs, and nucleotide derivatives. For example, the term includesnaturally occurring DNA or RNA monomers, nucleotides with backbonemodifications such as peptide nucleic acid (PNA) (M. Egholm et al.,Nature 365:566 [1993]), phosphorothioate DNA, phosphorodithioate DNA,phosphoramidate DNA, aminde-linked DNA, MMI-linked DNA, 2′-O-methyl RNA,alpha-DNA and methylphosphonate DNA, nucleotides with sugarmodifications such as 2′-O-methyl RNA, 2′-fluoro RNA, 2′-amino RNA,2′-O-alkyl DNA, 2′-O-allyl DNA, 2′-O-alkynyl DNA, hexose DNA, pyranosylRNA, and anhydrohexitol DNA, and nucleotides having base modificationssuch as C-5 substituted pyrimidines (substituents including fluoro-,bromo- chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-,propynyl-, alkynyl-, thiazoyl-, imidazolyl-, pyridyl-), 7-deazapurineswith C-7 substituents including fluoro-, bromo-, chloro-, iodo-,methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-,imidazolyl-, pyridyl-), inosine and diaminopurine.

[0132] The term “base analog” as used herein refers to modified ornon-naturally occurring bases such as 7-deaza purines (e.g.,7-deaza-adenine and 7-deaza-guanine); bases modified, for example, toprovide altered interactions such as non-standard basepairing,including, but not limited to: IsoC, Iso G, and other modified bases andnucleotides described in U.S. Pat. Nos. 5,432,272; 6,001,983; 6,037,120;6,140,496; 5,912,340; 6,127,121 and 6,143,877, each of which isincorporated herein by reference in their entireties; heterocyclic baseanalogs based no the purine or pyrimidine ring systems, and otherheterocyclic bases. Nucleotide analogs include base analogs and comprisemodified forms of deoxyribonucleotides as well as ribonucleotides.

[0133] The term “polymorphic locus” is a locus present in a populationthat shows variation between members of the population (e.g., the mostcommon allele has a frequency of less than 0.95). In contrast, a“monomorphic locus” is a genetic locus at little or no variations seenbetween members of the population (generally taken to be a locus atwhich the most common allele exceeds a frequency of 0.95 in the genepool of the population).

[0134] The term “microorganism” as used herein means an organism toosmall to be observed with the unaided eye and includes, but is notlimited to bacteria, virus, protozoans, fungi, and ciliates.

[0135] The term “microbial gene sequences” refers to gene sequencesderived from a microorganism.

[0136] The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

[0137] The term “virus” refers to obligate, ultramicroscopic,intracellular parasites incapable of autonomous replication (i.e.,replication requires the use of the host cell's machinery).

[0138] The term “multi-drug resistant” or multiple-drug resistant”refers to a microorganism that is resistant to more than one of theantibiotics or antimicrobial agents used in the treatment of saidmicroorganism.

[0139] The term “sample” in the present specification and claims is usedin its broadest sense. On the one hand it is meant to include a specimenor culture (e.g., microbiological cultures). On the other hand, it ismeant to include both biological and environmental samples. A sample mayinclude a specimen of synthetic origin.

[0140] Biological samples may be animal, including human, fluid, solid(e.g., stool) or tissue, as well as liquid and solid food and feedproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Biological samples may be obtained from all ofthe various families of domestic animals, as well as feral or wildanimals, including, but not limited to, such animals as ungulates, bear,fish, lagamorphs, rodents, etc.

[0141] Environmental samples include environmental material such assurface matter, soil, water and industrial samples, as well as samplesobtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention.

[0142] The term “source of target nucleic acid” refers to any samplethat contains nucleic acids (RNA or DNA). Particularly preferred sourcesof target nucleic acids are biological samples including, but notlimited to blood, saliva, cerebral spinal fluid, pleural fluid, milk,lymph, sputum and semen.

[0143] An oligonucleotide is said to be present in “excess” relative toanother oligonucleotide (or target nucleic acid sequence) if thatoligonucleotide is present at a higher molar concentration that theother oligonucleotide (or target nucleic acid sequence). When anoligonucleotide such as a probe oligonucleotide is present in a cleavagereaction in excess relative to the concentration of the complementarytarget nucleic acid sequence, the reaction may be used to indicate theamount of the target nucleic acid present. Typically, when present inexcess, the probe oligonucleotide will be present at least a 100-foldmolar excess; typically at least 1 pmole of each probe oligonucleotidewould be used when the target nucleic acid sequence was present at about10 fmoles or less.

[0144] A sample “suspected of containing” a first and a second targetnucleic acid may contain either, both or neither target nucleic acidmolecule.

[0145] The term “charge-balanced” oligonucleotide refers to anoligonucleotide (the input oligonucleotide in a reaction) that has beenmodified such that the modified oligonucleotide bears a charge, suchthat when the modified oligonucleotide is either cleaved (i.e.,shortened) or elongated, a resulting product bears a charge differentfrom the input oligonucleotide (the “charge-unbalanced” oligonucleotide)thereby permitting separation of the input and reacted oligonucleotideson the basis of charge. The term “charge-balanced” does not imply thatthe modified or balanced oligonucleotide has a net neutral charge(although this can be the case). Charge-balancing refers to the designand modification of an oligonucleotide such that a specific reactionproduct generated from this input oligonucleotide can be separated onthe basis of charge from the input oligonucleotide.

[0146] For example, in an INVADER oligonucleotide-directed cleavageassay in which the probe oligonucleotide bears the sequence: 5′TTCTTTTCACCAGCGAGACGGG 3′ (i.e., SEQ ID NO:136 without the modifiedbases) and cleavage of the probe occurs between the second and thirdresidues, one possible charge-balanced version of this oligonucleotidewould be: 5′ Cy3-AminoT-Amino-TCTTTTCACCAGCGAGAC GGG 3′. This modifiedoligonucleotide bears a net negative charge. After cleavage, thefollowing oligonucleotides are generated: 5′ Cy3-AminoT-Amino-T 3′ and5′ CTTTTCACCAGCGAGACGGG 3′ (residues 3-22 of SEQ ID NO:136). 5′Cy3-AminoT-Amino-T 3′ bears a detectable moiety (the positively-chargedCy3 dye) and two amino-modified bases. The amino-modified bases and theCy3 dye contribute positive charges in excess of the negative chargescontributed by the phosphate groups and thus the 5′ Cy3-AminoT-Amino-T3′oligonucleotide has a net positive charge. The other, longer cleavagefragment, like the input probe, bears a net negative charge. Because the5′ Cy3-AminoT-Amino-T 3′ fragment is separable on the basis of chargefrom the input probe (the charge-balanced oligonucleotide), it isreferred to as a charge-unbalanced oligonucleotide. The longer cleavageproduct cannot be separated on the basis of charge from the inputoligonucleotide as both oligonucleotides bear a net negative charge;thus, the longer cleavage product is not a charge-unbalancedoligonucleotide.

[0147] The term “net neutral charge” when used in reference to anoligonucleotide, including modified oligonucleotides, indicates that thesum of the charges present (i.e., R—NH3+ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction or separation conditions is essentially zero.An oligonucleotide having a net neutral charge would not migrate in anelectrical field.

[0148] The term “net positive charge” when used in reference to anoligonucleotide, including modified oligonucleotides, indicates that thesum of the charges present (i.e., R—NH3+ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is +1 or greater. Anoligonucleotide having a net positive charge would migrate toward thenegative electrode in an electrical field.

[0149] The term “net negative charge” when used in reference to anoligonucleotide, including modified oligonucleotides, indicates that thesum of the charges present (i.e., R—NH3+ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is −1 or lower. An oligonucleotidehaving a net negative charge would migrate toward the positive electrodein an electrical field.

[0150] The term “polymerization means” or “polymerization agent” refersto any agent capable of facilitating the addition of nucleosidetriphosphates to an oligonucleotide. Preferred polymerization meanscomprise DNA and RNA polymerases.

[0151] The term “ligation means” or “ligation agent” refers to any agentcapable of facilitating the ligation (i.e., the formation of aphosphodiester bond between a 3′-OH and a 5′ P located at the termini oftwo strands of nucleic acid). Preferred ligation means comprise DNAligases and RNA ligases.

[0152] The term “reactant” is used herein in its broadest sense. Thereactant can comprise, for example, an enzymatic reactant, a chemicalreactant or light (e.g., ultraviolet light, particularly shortwavelength ultraviolet light is known to break oligonucleotide chains).Any agent capable of reacting with an oligonucleotide to either shorten(i.e., cleave) or elongate the oligonucleotide is encompassed within theterm “reactant.”

[0153] The term “adduct” is used herein in its broadest sense toindicate any compound or element that can be added to anoligonucleotide. An adduct may be charged (positively or negatively) ormay be charge-neutral. An adduct may be added to the oligonucleotide viacovalent or non-covalent linkages. Examples of adducts include, but arenot limited to, indodicarbocyanine dye amidites, amino-substitutednucleotides, ethidium bromide, ethidium homodimer,(1,3-propanediamino)propidium, (diethylenetriamino)propidium, thiazoleorange, (N-N′-tetramethyl-1,3-propanediamino)propyl thiazole orange,(N-N′-tetramethyl-1,2-ethanediamino)propyl thiazole orange, thiazoleorange-thiazole orange homodimer (TOTO), thiazole orange-thiazole blueheterodimer (TOTAB), thiazole orange-ethidium heterodimer 1 (TOED 1),thiazole orange-ethidium heterodimer 2 (TOED2) and fluorescein-ethidiumheterodimer (FED), psoralens, biotin, streptavidin, avidin, etc.

[0154] Where a first oligonucleotide is complementary to a region of atarget nucleic acid and a second oligonucleotide has complementary tothe same region (or a portion of this region) a “region of sequenceoverlap” exists along the target nucleic acid. The degree of overlapwill vary depending upon the nature of the complementarity (see, e.g.,region “X” in FIGS. 29 and 67 and the accompanying discussions).

[0155] As used herein, the term “purified” or “to purify” refers to theremoval of contaminants from a sample. For example, recombinant CLEAVASEnucleases are expressed in bacterial host cells and the nucleases arepurified by the removal of host cell proteins; the percent of theserecombinant nucleases is thereby increased in the sample.

[0156] The term “recombinant DNA molecule” as used herein refers to aDNA molecule that comprises of segments of DNA joined together by meansof molecular biological techniques.

[0157] The term “recombinant protein” or “recombinant polypeptide” asused herein refers to a protein molecule that is expressed from arecombinant DNA molecule.

[0158] As used herein the term “portion” when in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid (e.g., 4, 5, 6, .. . , n−1).

[0159] The term “nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin that may besingle or double stranded, and represent the sense or antisense strand.Similarly, “amino acid sequence” as used herein refers to peptide orprotein sequence.

[0160] The term “peptide nucleic acid” (“PNA”) as used herein refers toa molecule comprising bases or base analogs such as would be found innatural nucleic acid, but attached to a peptide backbone rather than thesugar-phosphate backbone typical of nucleic acids. The attachment of thebases to the peptide is such as to allow the bases to base pair withcomplementary bases of nucleic acid in a manner similar to that of anoligonucleotide. These small molecules, also designated anti geneagents, stop transcript elongation by binding to their complementarystrand of nucleic acid (Nielsen, et al. Anticancer Drug Des. 8:53 63[1993]).

[0161] As used herein, the terms “purified” or “substantially purified”refer to molecules, either nucleic or amino acid sequences, that areremoved from their natural environment, isolated or separated, and areat least 60% free, preferably 75% free, and most preferably 90% freefrom other components with which they are naturally associated. An“isolated polynucleotide” or “isolated oligonucleotide” is therefore asubstantially purified polynucleotide.

[0162] As used herein, the term “fusion protein” refers to a chimericprotein containing the protein of interest (e.g., CLEAVASE BN/thrombinnuclease and portions or fragments thereof) joined to an exogenousprotein fragment (the fusion partner which consists of a non CLEAVASEBN/thrombin nuclease protein). The fusion partner may enhance solubilityof recombinant chimeric protein (e.g., the CLEAVASE BN/thrombinnuclease) as expressed in a host cell, may provide an affinity tag(e.g., a his-tag) to allow purification of the recombinant fusionprotein from the host cell or culture supernatant, or both. If desired,the fusion protein may be removed from the protein of interest (e.g.,CLEAVASE BN/thrombin nuclease or fragments thereof) by a variety ofenzymatic or chemical means known to the art.

[0163] As used herein, the terms “chimeric protein” and “chimericalprotein” refer to a single protein molecule that comprises amino acidsequences portions derived from two or more parent proteins. Theseparent molecules may be from similar proteins from genetically distinctorigins, different proteins from a single organism, or differentproteins from different organisms. By way of example but not by way oflimitation, a chimeric structure-specific nuclease of the presentinvention may contain a mixture of amino acid sequences that have beenderived from FEN-1 genes from two or more of the organisms having suchgenes, combined to form a non-naturally occurring nuclease. The term“chimerical” as used herein is not intended to convey any particularproportion of contribution from the naturally occurring genes, nor limitthe manner in which the portions are combined. Any chimericstructure-specific nuclease constructs having cleavage activity asdetermined by the testing methods described herein are improved cleavageagents within the scope of the present invention.

[0164] The term “continuous strand of nucleic acid” as used herein ismeans a strand of nucleic acid that has a continuous, covalently linked,backbone structure, without nicks or other disruptions. The dispositionof the base portion of each nucleotide, whether base-paired,single-stranded or mismatched, is not an element in the definition of acontinuous strand. The backbone of the continuous strand is not limitedto the ribose-phosphate or deoxyribose-phosphate compositions that arefound in naturally occurring, unmodified nucleic acids. A nucleic acidof the present invention may comprise modifications in the structure ofthe backbone, including but not limited to phosphorothioate residues,phosphonate residues, 2′ substituted ribose residues (e.g., 2′-O-methylribose) and alternative sugar (e.g., arabinose) containing residues.

[0165] The term “continuous duplex” as used herein refers to a region ofdouble stranded nucleic acid in which there is no disruption in theprogression of basepairs within the duplex (i.e., the base pairs alongthe duplex are not distorted to accommodate a gap, bulge or mismatchwith the confines of the region of continuous duplex). As used hereinthe term refers only to the arrangement of the basepairs within theduplex, without implication of continuity in the backbone portion of thenucleic acid strand. Duplex nucleic acids with uninterruptedbasepairing, but with nicks in one or both strands are within thedefinition of a continuous duplex.

[0166] The term “duplex” refers to the state of nucleic acids in whichthe base portions of the nucleotides on one strand are bound throughhydrogen bonding the their complementary bases arrayed on a secondstrand. The condition of being in a duplex form reflects on the state ofthe bases of a nucleic acid. By virtue of base pairing, the strands ofnucleic acid also generally assume the tertiary structure of a doublehelix, having a major and a minor groove. The assumption of the helicalform is implicit in the act of becoming duplexed.

[0167] The term “duplex dependent protein binding” refers to the bindingof proteins to nucleic acid that is dependent on the nucleic acid beingin a duplex, or helical form.

[0168] The term “duplex dependent protein binding sites or regions” asused herein refers to discrete regions or sequences within a nucleicacid that are bound with particular affinity by specificduplex-dependent nucleic acid binding proteins. This is in contrast tothe generalized duplex-dependent binding of proteins that are notsite-specific, such as the histone proteins that bind chromatin withlittle reference to specific sequences or sites.

[0169] The term “protein-binding region” as used herein refers to anucleic acid region identified by a sequence or structure as binding toa particular protein or class of proteins. It is within the scope ofthis definition to include those regions that contain sufficient geneticinformation to allow identifications of the region by comparison toknown sequences, but which might not have the requisite structure foractual binding (e.g., a single strand of a duplex-depending nucleic acidbinding protein site). As used herein “protein binding region” excludesrestriction endonuclease binding regions.

[0170] The term “complete double stranded protein binding region” asused herein refers to the minimum region of continuous duplex requiredto allow binding or other activity of a duplex-dependent protein. Thisdefinition is intended to encompass the observation that some duplexdependent nucleic acid binding proteins can interact with full activitywith regions of duplex that may be shorter than a canonical proteinbinding region as observed in one or the other of the two singlestrands. In other words, one or more nucleotides in the region may beallowed to remain unpaired without suppressing binding. As used here in,the term “complete double stranded binding region” refers to the minimumsequence that will accommodate the binding function. Because some suchregions can tolerate non-duplex sequences in multiple places, althoughnot necessarily simultaneously, a single protein binding region mighthave several shorter sub-regions that, when duplexed, will be fullycompetent for protein binding.

[0171] The term “template” refers to a strand of nucleic acid on which acomplementary copy is built from nucleoside triphosphates through theactivity of a template-dependent nucleic acid polymerase. Within aduplex the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand.

[0172] The term “template-dependent RNA polymerase” refers to a nucleicacid polymerase that creates new RNA strands through the copying of atemplate strand as described above and which does not synthesize RNA inthe absence of a template. This is in contrast to the activity of thetemplate-independent nucleic acid polymerases that synthesize or extendnucleic acids without reference to a template, such as terminaldeoxynucleotidyl transferase, or Poly A polymerase.

[0173] The term “ARRESTOR molecule” refers to an agent added to orincluded in an invasive cleavage reaction in order to stop one or morereaction components from participating in a subsequent action orreaction. This may be done by sequestering or inactivating some reactioncomponent (e.g., by binding or base-pairing a nucleic acid component, orby binding to a protein component). The term “ARRESTOR oligonucleotide”refers to an oligonucleotide included in an invasive cleavage reactionin order to stop or arrest one or more aspects of any reaction (e.g.,the first reaction and/or any subsequent reactions or actions; it is notintended that the ARRESTOR oligonucleotide be limited to any particularreaction or reaction step). This may be done by sequestering somereaction component (e.g., base-pairing to another nucleic acid, orbinding to a protein component). However, it is not intended that theterm be so limited as to just situations in which a reaction componentis sequestered.

[0174] As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the assay etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materials.As used herein, the term “fragmented kit” refers to a delivery systemscomprising two or more separate containers that each contain asubportion of the total kit components. The containers may be deliveredto the intended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains oligonucleotides. The term “fragmented kit” isintended to encompass kits containing Analyte specific reagents (ASR's)regulated under section 520(e) of the Federal Food, Drug, and CosmeticAct, but are not limited thereto. Indeed, any delivery system comprisingtwo or more separate containers that each contains a subportion of thetotal kit components are included in the term “fragmented kit.” Incontrast, a “combined kit” refers to a delivery system containing all ofthe components of a reaction assay in a single container (e.g., in asingle box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

[0175] As used herein, the term “functional domain” refers to a region,or a part of a region, of a protein (e.g., an enzyme) that provides oneor more functional characteristic of the protein. For example, afunctional domain of an enzyme may provide, directly or indirectly, oneor more activities of the enzyme including, but not limited to,substrate binding capability and catalytic activity. A functional domainmay be characterized through mutation of one or more amino acids withinthe functional domain, wherein mutation of the amino acid(s) alters theassociated functionality (as measured empirically in an assay) therebyindicating the presence of a functional domain.

[0176] As used herein, the term “heterologous functional domain” refersto a protein functional domain that is not in its natural environment.For example, a heterologous functional domain includes a functionaldomain from one enzyme introduced into another enzyme. A heterologousfunctional domain also includes a functional domain native to a proteinthat has been altered in some way (e.g., mutated, added in multiplecopies, etc.). A heterologous functional domain may comprise a pluralityof contiguous amino acids or may include two or more distal amino acidsare amino acids fragments (e.g., two or more amino acids or fragmentswith intervening, non-heterologous, sequence). Heterologous functionaldomains are distinguished from endogenous functional domains in that theheterologous amino acid(s) are joined to or contain amino acid sequencesthat are not found naturally associated with the amino acid sequence innature or are associated with a portion of a protein not found innature.

[0177] As used herein, the term “altered functionality in a nucleic acidcleavage assay” refers to a characteristic of an enzyme that has beenaltered in some manner to differ from its natural state (e.g., to differfrom how it is found in nature). Alterations include, but are notlimited to, addition of a heterologous functional domain (e.g., throughmutation or through creation of chimerical proteins). In someembodiments, the altered characteristic of the enzyme may be one thatimproves the performance of an enzyme in a nucleic acid cleavage assay.Types of improvement include, but are not limited to, improved nucleaseactivity (e.g., improved rate of reaction), improved substrate binding(e.g., increased or decreased binding of certain nucleic acid species[e.g., RNA or DNA] that produces a desired outcome [e.g., greaterspecificity, improved substrate turnover, etc.]), and improvedbackground specificity (e.g., less undesired product is produced). Thepresent invention is not limited by the nucleic cleavage assay used totest improved functionality. However, in some preferred embodiments ofthe present invention, an invasive cleavage assay is used as the nucleicacid cleavage assay. In certain particularly preferred embodiments, aninvasive cleavage assay utilizing an RNA target is used as the nucleicacid cleavage assay.

[0178] As used herein, the terms “N-terminal” and “C-terminal” inreference to polypeptide sequences refer to regions of polypeptidesincluding portions of the N-terminal and C-terminal regions of thepolypeptide, respectively. A sequence that includes a portion of theN-terminal region of polypeptide includes amino acids predominantly fromthe N-terminal half of the polypeptide chain, but is not limited to suchsequences. For example, an N-terminal sequence may include an interiorportion of the polypeptide sequence including bases from both theN-terminal and C-terminal halves of the polypeptide. The same applies toC-terminal regions. N-terminal and C-terminal regions may, but need not,include the amino acid defining the ultimate N-terminal and C-terminalends of the polypeptide, respectively.

DESCRIPTION OF THE DRAWINGS

[0179]FIG. 1 shows a schematic representation of sequential invasivecleavage reactions. In step A, an upstream INVADER oligonucleotide and adownstream probe combine with a target nucleic acid strand to form acleavage structure. In step B, the portion of the cleaved signal probefrom A combines with a second target nucleic acid strand and a labeledsignal probe to form a second cleavage structure. In step C, cleavage ofthe labeled second cleavage structure yields a detectable signal.

[0180]FIG. 2 shows schematic representations of several examples ofinvasive cleavage structures comprising RNA target strands (SEQ IDNO:141). Panel A depicts an INVADER oligonucleotide (SEQ ID NO:142) andprobe (SEQ ID NO:143). Panel B depicts an INVADER oligonucleotide (SEQID NO:144) and probe (SEQ ID NO:143). Panel C depicts an INVADERoligonucleotide (SEQ ID NO:145) and probe (SEQ ID NO:145). Panel Ddepicts an INVADER oligonucleotide (SEQ ID NO:145) and probe (SEQ IDNO:146).

[0181]FIG. 3 shows schematic representations of two examples ofstructures that are not invasive cleavage structures labelled SEQ IDNOs:147-152.

[0182]FIG. 4 shows a schematic representation of a configuration ofinvasive cleavage that is useful for detection of target sequencevariations. In A, an invasive cleavage structure having overlap betweenthe two probes is formed, and the arrow indicates that it is cleavableby the enzymes of the present invention. In B, variation of the targetsequence removes a region of complementarity to the downstream probe andeliminates the overlap. The absence of an arrow in panel B indicates areduced rate of cleavage of this structure compared to that diagrammedin panel A.

[0183]FIG. 5 shows a diagram of the X-ray structure of a ternary complexof Klentaq1 with primer/template DNA in the polymerizing mode determinedby Li et al. (Li et al., Protein Sci., 7:1116 [1998]). Without intendingto represent precise borders between features of the physical form, theportions referred to in the text as the “fingers”, “thumb” and “palm”regions are loosely indicated by the circle, rectangle, and oval,respectively.

[0184]FIG. 6 shows a schematic diagram of the DNA polymerase gene fromThermus aquaticus. Restriction sites used in these studies are indicatedabove. The approximate regions encoding various structural or functionaldomains of the protein are indicated by double-headed arrows, below.

[0185]FIG. 7 shows a schematic diagram of the chimeric constructscomprising portions of the TaqPol gene and the TthPol gene. Open andshaded boxes denote TaqPol and TthPol sequences, respectively. Thenumbers correspond to the amino acid sequence of TaqPol. The 5′ nucleaseand polymerase domains of TaqPol and the palm, thumb, and fingersregions of the polymerase domain are indicated. The abbreviations forthe restrictions sites used for recombination are as follows: E, EcoRI;N, NotI; Bs, BstBI; D, NdeI; B, BamHI; and S, SalI.

[0186] FIGS. 8A-H shows a comparison of the nucleotide structure of thepolymerase genes isolated from Thermus aquaticus (SEQ ID NO:153),Thermus flavus (SEQ ID NO:154) and Thermus thermophilus (SEQ ID NO:155);the consensus sequence (SEQ ID NO:156) is shown at the top of each row.

[0187] FIGS. 9A-C shows a comparison of the amino acid sequence of thepolymerase isolated from Thermus aquaticus (SEQ ID NO:157), Thermusflavus (SEQ ID NO:158), and Thermus thermophilus (SEQ ID NO:1); theconsensus sequence (SEQ ID NO:159) is shown at the top of each row.

[0188]FIG. 10 shows the sequences and proposed structures of substratesfor the invasive signal amplification reaction with human IL-6 RNAtarget strand (SEQ ID NO:160) and upstream probe (SEQ ID NO:161). Thecleavage site of the downstream probe (SEQ ID NO:162) is indicated by anarrow. Sequence of the IL-6 DNA target strand (SEQ ID NO:163) is shownbelow.

[0189]FIG. 11 shows the image generated by a fluorescence imager showingthe products of invasive cleavage assays using the indicated enzymes,and the IL-6 substrate of FIG. 10 having either a DNA target strand (A)or an RNA target strand (B).

[0190]FIG. 12 compares the cycling cleavage activities of Taq DN RX HT,Tth DN RX HT, and Taq-Tth chimerical enzymes with IL-6 substrate havingan RNA target strand.

[0191]FIG. 13 shows a comparison of the amino acid sequences of theBstI-BamHI fragments of TaqPol (SEQ ID NO:164) and TthPol (SEQ IDNO:165). Pairs of similar amino acids are shaded with light gray.Aligned amino acids that have a charge difference are shaded with darkgray. The numbers correspond to the amino acid sequence of TaqPol. Aminoacids of TaqPol changed to the corresponding amino acids of TthPol bysite-directed mutagenesis are indicated by (+).

[0192]FIG. 14 compares the cycling cleavage activities of Taq DN RX HT,Taq-Tth chimerical enzymes, and chimerical enzymes having the indicatedadditional amino acid modifications, with IL-6 substrate having an RNAtarget strand.

[0193]FIG. 15 compares the cycling cleavage activities of Taq DN RX HT,Tth DN RX HT, and Taq DN RX HT having the indicated amino acidmodifications, with IL-6 substrate having an RNA target strand.

[0194]FIG. 16 compares polymerization activities of TaqPol, TthPol, andTaq-Tth chimerical enzymes, and TaqPol having the indicated amino acidmodifications.

[0195]FIG. 17 shows a diagram of the X-ray structure of a ternarycomplex of Klentaq1 with primer/template DNA in the polymerizing modedetermined by Li et al. (Li et al., Protein Sci., 7:1116 [1998]). Aminoacids G418 and E507 are indicated.

[0196] FIGS. 18A-D show schematic diagrams of examples of substratesthat may be used to measure various cleavage activities of enzymes. Thesubstrates may be labeled, for example, with a fluorescent dye and aquenching moiety for FRET detection, as shown, to facilitate detectionand measurement. The substrates of 18A and 18B are invasive cleavagestructures having RNA and DNA target strands, respectively. 18C shows anexample of an X-structure, and 18D shows an example of a hairpinstructure, both of which may be used to assess the activity of enzymeson alternative structures that may be present in invasive cleavagereactions.

[0197]FIG. 19 shows schematic diagrams of chimeric constructs comprisingportions of the TaqPol gene and the TthPol gene. Open and shaded boxesdenote TaqPol and TthPol sequences, respectively. The chimeras alsoinclude the DN, RX, and HT modifications. A table compares the cleavageactivity of each protein on the indicated cleavage substrates.

[0198]FIG. 20A shows a schematic diagram for an RNA containing invasivecleavage substrate. The 5′ end of the target molecule (SEQ ID NO:166) ismodified with biotin and blocked with streptavidin as described. Thedownstream probe (SEQ ID NO:167) with cleavage site is also shown.Panels B-D show analysis of the properties of the Taq DN RX HTG418K/E507Q mutant in cleavage of the shown substrate under conditionsof varying reaction temperature, KCl concentration, and MgSO₄concentration.

[0199]FIG. 21 shows schematic diagrams for model substrates used to testenzymes for invasive cleavage activity. The molecule shown in 21Aprovides a DNA target strand (SEQ ID NO:168), while the model shown in21B provides an RNA containing target strand (SEQ ID NO:167). Both 21Aand B show downstream probe SEQ ID NO:166.

[0200]FIG. 22 shows schematic diagrams for model substrates used to testenzymes for cleavage activity on alternative, non-invasive structures.

[0201]FIG. 23 shows a schematic diagram for a model substrate used totest enzymes for invasive cleavage activity.

[0202]FIG. 24 shows schematic diagrams for a model substrate used totest enzymes for invasive cleavage activity on RNA or DNA targetstrands.

[0203]FIG. 25 compares the cycling cleavage activities of Tth DN RX HT,Taq 2M, TfiPol, Tsc Pol, and Tfi and Tsc-derived mutant enzymes.

[0204]FIG. 26 depicts structures that may be employed to determine theablity of an enzyme to cleave a probe in the presence and the absence ofan upstream oligonucleotide.

[0205]FIG. 26 displays the sequence of oligonucleotide 89-15-1 (SEQ IDNO:212), oligonucleotide 81-69-5 (SEQ ID NO:213), oligonucleotide81-69-4 (SEQ ID NO:214), oligonucleotide 81-69-3 (SEQ ID NO:215),oligonucleotide 81-69-2 (SEQ ID NO:216) and a portion of M13 mp18 (SEQID NO:217).

[0206]FIG. 27 shows the image generated by a fluorescence imager thatshows the dependence of Pfu FEN-1 on the presence of an overlappingupstream oligonucleotide for specific cleavage of the probe.

[0207]FIG. 28a shows the image generated by a fluorescence imager thatcompares the amount of product generated in a standard (i.e., anon-sequential invasive cleavage reaction) and a sequential invasivecleavage reaction.

[0208]FIG. 28b is a graph comparing the amount of product generated in astandard or basic (i.e., a non-sequential invasive cleavage reaction)and a sequential invasive cleavage reaction (“INVADER sqrd”) (yaxis=fluorescence units; x axix=attomoles of target).

[0209]FIG. 29 shows the image generated by a fluorescence imager thatshows that the products of a completed sequential invasive cleavagereaction cannot cross contaminant a subsequent similar reaction.

[0210]FIG. 30 shows the sequence of the oligonucleotide employed in aninvasive cleavage reaction for the detection of HCMV viral DNA; FIG. 30shows the sequence of oligonucleotide 89-76 (SEQ ID NO:218),oligonucleotide 89-44 (SEQ ID NO:219) and nucleotides 3057-3110 of theHCMV genome (SEQ ID NO:220).

[0211]FIG. 31 shows the image generated by a fluorescence imager thatshows the sensitive detection of HCMV viral DNA in samples containinghuman genomic DNA using an invasive cleavage reaction.

[0212]FIG. 32 is a schematic that illustrates one embodiment of thepresent invention, where the cut probe from an initial invasive cleavagereaction is employed as the INVADERoligonucleotide in a second invasivecleavage reaction, and where an ARRESTOR oligonucleotide preventsparticipation of remaining uncut first probe in the cleavage of thesecond probe.

[0213]FIG. 33 is a schematic that illustrates one embodiment of thepresent invention, where the cut probe from an initial invasive cleavagereaction is employed as an integrated INVADER-target complex in a secondinvasive cleavage reaction, and where an ARRESTOR oligonucleotideprevents participation of remaining uncut first probe in the cleavage ofthe second probe.

[0214]FIG. 34 shows three images generated by a fluorescence imagershowing that two different lengths of 2′ O-methyl, 3′ terminalamine-modified ARRESTOR oligonucleotide both reduce non-specificbackground cleavage of the secondary probe when included in the secondstep of a reaction where the cut probe from an initial invasive cleavagereaction is employed as an integrated INVADER-target complex in a secondinvasive cleavage reaction.

[0215]FIG. 35A shows two images generated by a fluorescence imagershowing the effects on nonspecific and specific cleavage signal ofincreasing concentrations of primary probe in the first step of areaction where the cut probe from an initial invasive cleavage reactionis employed as the INVADER oligonucleotide in a second invasive cleavagereaction.

[0216]FIG. 35B shows two images generated by a fluorescence imagershowing the effects on nonspecific and specific cleavage signal ofincreasing concentrations of primary probe in the first step of areaction, and inclusion of a 2′ O-methyl, 3′ terminal amine-modifiedARRESTOR oligonucleotide in the second step of a reaction where the cutprobe from an initial invasive cleavage reaction is employed as theINVADER oligonucleotide in a second invasive cleavage reaction.

[0217]FIG. 35C shows shows a graph generated using the spreadsheetMICROSOFT EXCEL software, comparing the effects on nonspecific andspecific cleavage signal of increasing concentrations of primary probein the first step of a reaction, in the presence or absence of a 2′O-methyl, 3′ terminal amine-modified ARRESTOR oligonucleotide in thesecond step of a reaction where the cut probe from an initial invasivecleavage reaction is employed as the INVADER oligonucleotide in a secondinvasive cleavage reaction.

[0218]FIG. 36A shows two images generated by a fluorescence imagershowing the effects on nonspecific and specific cleavage signal ofincluding an unmodified ARRESTOR oligonucleotide in the second step of areaction where the cut probe from an initial invasive cleavage reactionis employed as the INVADER oligonucleotide in a second invasive cleavagereaction.

[0219]FIG. 36B shows two images generated by a fluorescence imagershowing the effects on nonspecific and specific cleavage signal ofincluding a 3′ terminal amine modified ARRESTOR oligonucleotide, apartially 2′ O-methyl substituted, 3′ terminal amine modified ARRESTORoligonucleotide, or an entirely 2′ O-methyl, 3′ terminal amine modifiedARRESTOR oligonucleotide in the second step of a reaction where the cutprobe from an initial invasive cleavage reaction is employed as theINVADER oligonucleotide in a second invasive cleavage reaction.

[0220]FIG. 37A shows two images generated by a fluorescence imagercomparing the effects on nonspecific and specific cleavage signal ofincluding ARRESTOR oligonucleotides of different lengths in the secondstep of a reaction where the cut probe from an initial invasive cleavagereaction is employed as the INVADER oligonucleotide in a second invasivecleavage reaction.

[0221]FIG. 37B shows two images generated by a fluorescence imagercomparing the effects on nonspecific and specific cleavage signal ofincluding an arrestoer oligonucleotides of different lengths in thesecond step of a reaction where the cut probe from an initial invasivecleavage reaction is employed as the INVADER oligonucleotide in a secondinvasive cleavage reaction, and in which a longer variant of thesecondary probe used in the reactions in FIG. 37A is tested.

[0222]FIG. 37C shows a schematic diagram of a primary probe aligned withseveral ARRESTOR oligonucleotides of different lengths. The region ofthe primary probe that is complementary to the HBV target sequence isunderlined. The ARRESTOR oligonucleotides are aligned with the probe bycomplementarity.

[0223]FIG. 38 shows two images generated by a fluorescence imagercomparing the effects on nonspecific and specific cleavage signal ofincluding ARRESTOR oligonucleotides of different lengths in the secondstep of a reaction where the cut probe from an initial invasive cleavagereaction is employed as the INVADER oligonucleotide in a second invasivecleavage reaction, using secondary probes of two different lengths.

[0224]FIG. 39 shows a graph of the calculated running average of a tennucleotide stretch of the hUbiquitin RNA (the Ave(10) Index) derivedfrom the SS-count output of an mfold analysis, expressed as a percentageof the total number of structures found by mfold that include aparticular base, plotted against the position of the base.

[0225]FIG. 40 shows an example microplate layout for an RNA INVADERassay comprising 40 samples, 6 standards, and a No Target Control.

[0226]FIG. 41 shows INVADER assay components for use in detecting human(h), mouse (m), or rat (r) RNAs of the indicated genes or transcripts.

[0227]FIG. 42 shows a computer display of an INVADERCREATOR Order Entryscreen.

[0228]FIG. 43 shows a computer display of an INVADERCREATOR Multiple SNPDesign Selection screen.

[0229]FIG. 44 shows a computer display of an INVADERCREATOR DesignerWorksheet screen.

[0230]FIG. 45 shows a computer display of an INVADERCREATOR Output Pagescreen.

[0231]FIG. 46 shows a computer display of an INVADERCREATOR PrinterReady Output screen.

[0232]FIG. 47 shows INVADER assay components (SEQ ID NOs:709-2640) foruse in detecting RNA target nucleic acids. These assay components canalso be used in the detection of cDNA or DNA copies of the RNA targets.Components are grouped per RNA analyte to be detected. Where multipleprobes, INVADER oligonucleotides, stacker oligonucleotides, ARRESTORoligonucleotides, or other components are provided, any of the multiplecomponents may be used, unless indicated otherwise. Unless indicatedotherwise, oligonucleotides are presented 5′-3′ orientation.

[0233]FIG. 48 shows a chart showing the Ave(10) Index against base pairposition.

[0234]FIG. 49 shows INVADER assay components (SEQ ID NOs:2868-4004) foruse in detecting RNA target nucleic acids. These assay components canalso be used in the detection of cDNA or DNA copies of the RNA targets.Components are grouped per RNA analyte to be detected. Where multipleprobes, INVADER oligonucleotides, stacker oligonucleotides, ARRESTORoligonucleotides, or other components are provided, any of the multiplecomponents may be used, unless indicated otherwise. Unless indicatedotherwise, oligonucleotides are presented 5′-3′ orientation. All primaryprobes (i.e., oligonucleotides indicated as “Probes” that are not partof a Secondary System) include a 3′ amine (this may or may not beindicated), unless another chemistry is specifically identified. Naturalbases are designated with upper case characters, and 2′ O-methylatedbases are designated with lower case characters.

[0235]FIGS. 50A and B show schematic diagrams of two INVADER assayembodiments.

DESCRIPTION OF THE INVENTION

[0236] Introduction

[0237] The present invention relates to methods and compositions fortreating nucleic acid, and in particular, methods and compositions fordetection and characterization of nucleic acid sequences and sequencechanges.

[0238] In preferred embodiments, the present invention relates to meansfor cleaving a nucleic acid cleavage structure in a site-specificmanner. While the present invention provides a variety of cleavageagents, in some embodiments, the present invention relates to a cleavingenzyme having 5′ nuclease activity without interfering nucleic acidsynthetic ability. In other embodiments, the present invention providesnovel polymerases (e.g., thermostable polymerases) possessing alteredpolymerase and/or nucleases activities.

[0239] For example, in some embodiments, the present invention provides5′ nucleases derived from thermostable DNA polymerases that exhibitaltered DNA synthetic activity from that of native thermostable DNApolymerases. The 5′ nuclease activity of the polymerase is retainedwhile the synthetic activity is reduced or absent. Such 5′ nucleases arecapable of catalyzing the structure-specific cleavage of nucleic acidsin the absence of interfering synthetic activity. The lack of syntheticactivity during a cleavage reaction results in nucleic acid cleavageproducts of uniform size.

[0240] The novel properties of the nucleases of the invention form thebasis of a method of detecting specific nucleic acid sequences. Thismethod relies upon the amplification of the detection molecule ratherthan upon the amplification of the target sequence itself as do existingmethods of detecting specific target sequences.

[0241] DNA polymerases (DNAPs), such as those isolated from E. coli orfrom thermophilic bacteria of the genus Thermus as well as otherorganisms, are enzymes that synthesize new DNA strands. Several of theknown DNAPs contain associated nuclease activities in addition to thesynthetic activity of the enzyme.

[0242] Some DNAPs are known to remove nucleotides from the 5′ and 3′ends of DNA chains (Kornberg, DNA Replication, W. H. Freeman and Co.,San Francisco, pp. 127-139 [1980]). These nuclease activities areusually referred to as 5′ exonuclease and 3′ exonuclease activities,respectively. For example, the 5′ exonuclease activity located in theN-terminal domain of several DNAPs participates in the removal of RNAprimers during lagging strand synthesis during DNA replication and theremoval of damaged nucleotides during repair. Some DNAPs, such as the E.coli DNA polymerase (DNAPEc1), also have a 3′ exonuclease activityresponsible for proof-reading during DNA synthesis (Kornberg, supra).

[0243] A DNAP isolated from Thermus aquaticus, termed Taq DNA polymerase(DNAPTaq), has a 5′ exonuclease activity, but lacks a functional 3′exonucleolytic domain (Tindall and Kunkell, Biochem., 27:6008 [1988]).Derivatives of DNAPEc1 and DNAPTaq, respectively called the Klenow andStoffel fragments, lack 5′ exonuclease domains as a result of enzymaticor genetic manipulations (Brutlag et al., Biochem. Biophys. Res.Commun., 37:982 [1969]; Erlich et al., Science 252:1643 [1991]; Setlowand Komberg, J. Biol. Chem., 247:232 [1972]).

[0244] The 5′ exonuclease activity of DNAPTaq was reported to requireconcurrent synthesis (Gelfand, PCR Technology—Principles andApplications for DNA Amplification, H. A. Erlich, [Ed.], Stockton Press,New York, p. 19 [1989]). Although mononucleotides predominate among thedigestion products of the 5′ exonucleases of DNAPTaq and DNAPEc1, shortoligonucleotides (≦12 nucleotides) can also be observed implying thatthese so-called 5′ exonucleases can function endonucleolytically(Setlow, supra; Holland et al., Proc. Natl. Acad. Sci. USA 88:7276[1991]).

[0245] In WO 92/06200, Gelfand et al. show that the preferred substrateof the 5′ exonuclease activity of the thermostable DNA polymerases isdisplaced single-stranded DNA. Hydrolysis of the phosphodiester bondoccurs between the displaced single-stranded DNA and the double-helicalDNA with the preferred exonuclease cleavage site being a phosphodiesterbond in the double helical region. Thus, the 5′ exonuclease activityusually associated with DNAPs is a structure-dependent single-strandedendonuclease and is more properly referred to as a 5′ nuclease.Exonucleases are enzymes that cleave nucleotide molecules from the endsof the nucleic acid molecule. Endonucleases, on the other hand, areenzymes that cleave the nucleic acid molecule at internal rather thanterminal sites. The nuclease activity associated with some thermostableDNA polymerases cleaves endonucleolytically but this cleavage requirescontact with the 5′ end of the molecule being cleaved. Therefore, thesenucleases are referred to as 5′ nucleases.

[0246] When a 5′ nuclease activity is associated with a eubacterial TypeA DNA polymerase, it is found in the one third N-terminal region of theprotein as an independent functional domain. The C-terminal two-thirdsof the molecule constitute the polymerization domain that is responsiblefor the synthesis of DNA. Some Type A DNA polymerases also have a 3′exonuclease activity associated with the two-third C-terminal region ofthe molecule.

[0247] The 5′ exonuclease activity and the polymerization activity ofDNAPs can be separated by proteolytic cleavage or genetic manipulationof the polymerase molecule. The Klenow or large proteolytic cleavagefragment of DNAPEc1 contains the polymerase and 3′ exonuclease activitybut lacks the 5′ nuclease activity. The Stoffe1 fragment of DNAPTaq(DNAPStf) lacks the 5′ nuclease activity due to a genetic manipulationthat deleted the N-terminal 289 amino acids of the polymerase molecule(Erlich et al., Science 252:1643 [1991]). WO 92/06200 describes athermostable DNAP with an altered level of 5′ to 3′ exonuclease. U.S.Pat. No. 5,108,892 describes a Thermus aquaticus DNAP without a 5′ to 3′exonuclease. Thermostable DNA polymerases with lessened amounts ofsynthetic activity are available (Third Wave Technologies, Madison,Wis.) and are described in U.S. Pat. Nos. 5,541,311, 5,614,402,5,795,763, 5,691,142, and 5,837,450, herein incorporated by reference intheir entireties. The present invention provides 5′ nucleases derivedfrom thermostable Type A DNA polymerases that retain 5′ nucleaseactivity but have reduced or absent synthetic activity. The ability touncouple the synthetic activity of the enzyme from the 5′ nucleaseactivity proves that the 5′ nuclease activity does not requireconcurrent DNA synthesis as was previously reported (Gelfand, PCRTechnology, supra).

[0248] In addition to the 5′-exonuclease domains of the DNA polymerase Iproteins of Eubacteria, described above, 5′ nucleases have been foundassociated with bacteriophage, eukaryotes and archaebacteria. Overall,all of the enzymes in this family display very similar substratespecificities, despite their limited level of sequence similarity.Consequently, enzymes suitable for use in the methods of the presentinvention may be isolated or derived from a wide array of sources.

[0249] A mammalian enzyme with functional similarity to the5′-exonuclease domain of E. coli Pol I was isolated nearly 30 years ago(Lindahl, et al., Proc Natl Acad Sci USA 62(2): 597-603 [1969]). Later,additional members of this group of enzymes called flap endonucleases(FEN1) from Eukarya and Archaea were shown to possess a nearly identicalstructure specific activity (Harrington and Lieber. Embo J 13(5),1235-46 [1994]; Murante et al., J Biol Chem 269(2), 1191-6 [1994];Robins, et al., J Biol Chem 269(46), 28535-8 [1994]; Hosfield, et al., JBiol Chem 273(42), 27154-61 [1998]), despite limited sequencesimilarity. The substrate specificities of the FEN1 enzymes, and theeubacterial and related bacteriophage enzymes have been examined andfound to be similar for all enzymes (Lyamichev, et al., Science260(5109), 778-83 [1993], Harrington and Lieber, supra, Murante, et al.,supra, Hosfield, et al, supra, Rao, et al., J Bacteriol 180(20), 5406-12[1998], Bhagwat, et al., J. Biol Chem 272(45), 28523-30 [1997], Garforthand Sayers, Nucleic Acids Res 25(19), 3801-7 [1997]).

[0250] Using preformed substrates, many of the studies cited abovedetermined that these nucleases leave a gap upon cleavage, leading theauthors to speculate that DNA polymerase must then act to fill in thatgap to generate a ligatable nick. A number of other 5′ nucleases havebeen shown to leave a gap or overlap after cleavage of the same orsimilar flap substrates. It has since been determined that that all thestructure-specific 5′-exonucleases leave a nick after cleavage if thesubstrate has an overlap between the upstream and downstream duplexes(Kaiser et al., J. Biol. Chem. 274(30):21387-21394 [1999]). Whileduplexes having several bases of overlapping sequence can assume severaldifferent conformations through branch migration, it was determined thatcleavage occurs in the conformation where the last nucleotide at the 3′end of the upstream strand is unpaired, with the cleavage rate beingessentially the same whether the end of the upstream primer is A, C, G,or T. It was determined to be positional overlap between the 3′ end ofthe upstream primer and downstream duplex, rather then sequence overlap,that provides optimal cleavage. In addition to allowing these enzymes toleave a nick after cleavage, the single base of overlap causes theenzymes to cleave several orders of magnitude faster than when asubstrate lacks overlap (Kaiser et al., supra).

[0251] Any of the 5′ nucleases described below may find application inone or more embodiments of the methods described herein. 5′ nucleases ofparticular utility in the methods of present invention include but arenot limited to polymerases from a Thermus species including, but notlimited to, Thermus aquaticus, Thermus flavus, Thermus thermophilus,Thermus filiformus, and Thermus scotoductus, and altered polymerases.Particularly preferred are altered polymerases exhibiting improvedperformance in detection assays based on the cleavage of a DNA member ofan invasive cleavage structure that comprises an RNA target strand.

[0252] Chimerical polymerases may find application in one or moreembodiments of the present invention, including but not limited tochimerical polymerases comprising one or more portions of one or moreFEN nucleases including but are not limited to those of Methanococcusjannaschii, Methanobacterium thermoautotrophicum, Archaeoglobusveneficus, Sulfolobus solfataricus, Pyrobaculum aerophilum, Thermococcuslitoralis, Archaeaglobus profundus, Acidianus brierlyi, Acidianusambivalens, Desulfurococcus amylolyticus, Desulfurococcus mobilis,Pyrodictium brockii, Thermococcus gorgonarius, Thermococcus zilligii,Methanopyrus kandleri, Methanococcus igneus, Pyrococcus horikoshii, andAeropyrum pernix; particularly preferred FEN1 enzymes are chimericalArchaeoglobus fulgidus and Pyrococcus furiosus. Particularly preferredare altered polymerases exhibiting improved performance in detectionassays based on the cleavage of a DNA member of an invasive cleavagestructure that comprises an RNA target strand.

[0253] The detailed description of the invention is presented in thefollowing sections:

[0254] I. Detection of Specific Nucleic Acid Sequences Using 5′Nucleases in an INVADER Directed Cleavage Assay;

[0255] II. Signal Enhancement By Incorporating The Products Of AnInvasive Cleavage Reaction Into A Subsequent Invasive Cleavage Reaction;

[0256] III. Effect of ARRESTOR Oligonucleotides on Signal and Backgroundin Sequential Invasive Cleavage Reactions.

[0257] IV. Improved Enzymes For Use In INVADER Oligonucleotide-DirectedCleavage Reactions Comprising RNA Targets;

[0258] V. Reaction Design for INVADER Assay Detection of RNA Targets;

[0259] VI. Kits for performing the RNA INVADER Assay; and

[0260] VII. The INVADER Assay for Direct Detection and Measurement ofSpecific RNA Analytes.

[0261] I. Detection of Specific Nucleic Acid Sequences Using 5′Nucleases in an INVADER Directed Cleavage Assay

[0262] 1. INVADER Assay Reaction Design

[0263] The present invention provides means for forming a nucleic acidcleavage structure that is dependent upon the presence of a targetnucleic acid and cleaving the nucleic acid cleavage structure so as torelease distinctive cleavage products. 5′ nuclease activity, forexample, is used to cleave the target-dependent cleavage structure andthe resulting cleavage products are indicative of the presence ofspecific target nucleic acid sequences in the sample. When two strandsof nucleic acid, or oligonucleotides, both hybridize to a target nucleicacid strand such that they form an overlapping invasive cleavagestructure, as described below, invasive cleavage can occur. Through theinteraction of a cleavage agent (e.g., a 5′ nuclease) and the upstreamoligonucleotide, the cleavage agent can be made to cleave the downstreamoligonucleotide at an internal site in such a way that a distinctivefragment is produced. Such embodiments have been termed the INVADERassay (Third Wave Technologies) and are described in U.S. Pat. Nos.5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,348,314,and PCT Publications WO 97/27214 and WO 98/42873, herein incorporated byreference in their entireties.

[0264] The present invention further provides assays in which the targetnucleic acid is reused or recycled during multiple rounds ofhybridization with oligonucleotide probes and cleavage of the probeswithout the need to use temperature cycling (i.e., for periodicdenaturation of target nucleic acid strands) or nucleic acid synthesis(i.e., for the polymerization-based displacement of target or probenucleic acid strands). When a cleavage reaction is run under conditionsin which the probes are continuously replaced on the target strand (e.g.through probe-probe displacement or through an equilibrium betweenprobe/target association and disassociation, or through a combinationcomprising these mechanisms, [Reynaldo et al., J. Mol. Biol. 97: 511(2000)]) multiple probes can hybridize to the same target, allowingmultiple cleavages, and the generation of multiple cleavage products.

[0265] By the extent of its complementarity to a target nucleic acidstrand, an oligonucleotide may be said to define a specific region ofthe target. In an invasive cleavage structure, the two oligonucleotidesdefine and hybridize to regions of the target that are adjacent to oneanother (i.e., regions without any additional region of the targetbetween them). Either or both oligonucleotides may comprise additionalportions that are not complementary to the target strand. In addition tohybridizing adjacently, in order to form an invasive cleavage structure,the 3′ end of the upstream oligonucleotide must comprise an additionalmoiety. When both oligonucleotides are hybridized to a target strand toform a structure and such a 3′ moiety is present on the upstreamoligonucleotide within the structure, the oligonucleotides may be saidto overlap, and the structure may be described as an overlapping, orinvasive cleavage structure.

[0266] In one embodiment, the 3′ moiety of the invasive cleavagestructure is a single nucleotide. In this embodiment the 3′ moiety maybe any nucleotide (i.e., it may be, but it need not be complementary tothe target strand). In a preferred embodiment the 3′ moiety is a singlenucleotide that is not complementary to the target strand. In anotherembodiment, the 3′ moiety is a nucleotide-like compound (i.e., a moietyhaving chemical features similar to a nucleotide, such as a nucleotideanalog or an organic ring compound; See e.g., U.S. Pat. No. 5,985,557).In yet another embodiment the 3′ moiety is one or more nucleotides thatduplicate in sequence one or more nucleotides present at the 5′ end ofthe hybridized region of the downstream oligonucleotide. In a furtherembodiment, the duplicated sequence of nucleotides of the 3′ moiety isfollowed by a single nucleotide that is not further duplicative of thedownstream oligonucleotide sequence, and that may be any othernucleotide. In yet another embodiment, the duplicated sequence ofnucleotides of the 3′ moiety is followed by a nucleotide-like compound,as described above.

[0267] The downstream oligonucleotide may have, but need not have,additional moieties attached to either end of the region that hybridizesto the target nucleic acid strand. In a preferred embodiment, thedownstream oligonucleotide comprises a moiety at its 5′ end (i.e., a 5′moiety). In a particularly preferred embodiment, said 5′ moiety is a 5′flap or arm comprising a sequence of nucleotides that is notcomplementary to the target nucleic acid strand.

[0268] When an overlapping cleavage structure is formed, it can berecognized and cleaved by a nuclease that is specific for this structure(i.e., a nuclease that will cleave one or more of the nucleic acids inthe overlapping structure based on recognition of this structure, ratherthan on recognition of a nucleotide sequence of any of the nucleic acidsforming the structure). Such a nuclease may be termed a“structure-specific nuclease”. In some embodiments, thestructure-specific nuclease is a 5′ nuclease. In a preferred embodiment,the structure-specific nuclease is the 5′ nuclease of a DNA polymerase.In another preferred embodiment, the DNA polymerase having the 5′nuclease is synthesis-deficient. In another preferred embodiment, the 5′nuclease is a FEN-1 endonuclease. In a particularly preferredembodiment, the 5′ nuclease is thermostable.

[0269] In some embodiments, said structure-specific nucleasepreferentially cleaves the downstream oligonucleotide. In a preferredembodiment, the downstream oligonucleotide is cleaved one nucleotideinto the 5′ end of the region that is hybridized to the target withinthe overlapping structure. Cleavage of the overlapping structure at anylocation by a structure-specific nuclease produces one or more releasedportions or fragments of nucleic acid, termed “cleavage products.”

[0270] In some embodiments, cleavage of an overlapping structure isperformed under conditions wherein one or more of the nucleic acids inthe structure can disassociate (i.e. un-hybridize, or melt) from thestructure. In one embodiment, full or partial disassociation of a firstcleavage structure allows the target nucleic acid to participate in theformation of one or more additional overlapping cleavage structures. Ina preferred embodiment, the first cleavage structure is partiallydisassociated. In a particularly preferred embodiment only theoligonucleotide that is cleaved disassociates from the first cleavagestructure, such that it may be replaced by another copy of the sameoligonucleotide. In some embodiments, said disassociation is induced byan increase in temperature, such that one or more oligonucleotides canno longer hybridize to the target strand. In other embodiments, saiddisassociation occurs because cleavage of an oligonucleotide producesonly cleavage products that cannot bind to the target strand under theconditions of the reaction. In a preferred embodiment, conditions areselected wherein an oligonucleotide may associate with (i.e., hybridizeto) and disassociate from a target strand regardless of cleavage, andwherein the oligonucleotide may be cleaved when it is hybridized to thetarget as part of an overlapping cleavage structure. In a particularlypreferred embodiment, conditions are selected such that the number ofcopies of the oligonucleotide that can be cleaved when part of anoverlapping structure exceeds the number of copies of the target nucleicacid strand by a sufficient amount that when the first cleavagestructure disassociates, the probability that the target strand willassociate with an intact copy of the oligonucleotide is greater than theprobability that that it will associate with a cleaved copy of theoligonucleotide.

[0271] In some embodiments, cleavage is performed by astructure-specific nuclease that can recognize and cleave structuresthat do not have an overlap. In a preferred embodiment, cleavage isperformed by a structure-specific nuclease having a lower rate ofcleavage of nucleic acid structures that do not comprise an overlap,compared to the rate of cleavage of structures comprising an overlap. Ina particularly preferred embodiment, cleavage is performed by astructure-specific nuclease having less than 1% of the rate of cleavageof nucleic acid structures that do not comprise an overlap, compared tothe rate of cleavage of structures comprising an overlap.

[0272] In some embodiments it is desirable to detect the cleavage of theoverlapping cleavage structure. Detection may be by analysis of cleavageproducts or by analysis of one or more of the remaining uncleavednucleic acids. For convenience, the following discussion will refer tothe analysis of cleavage products, but it will be appreciated by thoseskilled in the art that these methods may as easily be applied toanalysis of the uncleaved nucleic acids in an invasive cleavagereaction. Any method known in the art for analysis of nucleic acids,nucleic acid fragments or oligonucleotides may be applied to thedetection of cleavage products.

[0273] In one embodiment, the cleavage products may be identified bychemical content, e.g., the relative amounts of each atom, eachparticular type of reactive group or each nucleotide base (Chargaff etal., J. Biol. Chem. 177: 405 [1949]) they contain. In this way, acleavage product may be distinguished from a longer nucleic acid fromwhich it was released by cleavage, or from other nucleic acids.

[0274] In another embodiment, the cleavage products may be distinguishedby a particular physical attribute, including but not limited to length,mass, charge, or charge-to-mass ratio. In yet another embodiment, thecleavage product may be distinguished by a behavior that is related to aphysical attribute, including but not limited to rate of rotation insolution, rate of migration during electrophoresis, coefficient ofsedimentation in centrifugation, time of flight in MALDI-TOF massspectrometry, migration rate or other behavior in chromatography,melting temperature from a complementary nucleic acid, orprecipitability from solution.

[0275] Detection of the cleavage products may be through release of alabel. Such labels may include, but are not limited to one or more ofany of dyes, radiolabels such as ³²P or ³⁵S, binding moieties such asbiotin, mass tags, such as metal ions or chemical groups, charge tags,such as polyamines or charged dyes, haptens such as digoxgenin,luminogenic, phosphorescent or fluorogenic moieties, and fluorescentdyes, either alone or in combination with moieties that can suppress orshift emission spectra, such as by fluorescence resonance energytransfer (FRET) or collisional fluorescence energy transfer.

[0276] In some embodiments, analysis of cleavage products may includephysical resolution or separation, for example by electrophoresis,hybridization or by selective binding to a support, or by massspectrometry methods such as MALDI-TOF. In other embodiments, theanalysis may be performed without any physical resolution or separation,such as by detection of cleavage-induced changes in fluorescence as inFRET-based analysis, or by cleavage-induced changes in the rotation rateof a nucleic acid in solution as in fluorescence polarization analysis.

[0277] Cleavage products can be used subsequently in any reaction orread-out method that can make use of oligonucleotides. Such reactionsinclude, but are not limited to, modification reactions, such asligation, tailing with a template-independent nucleic acid polymeraseand primer extension with a template-dependent nucleic acid polymerase.The modification of the cleavage products may be for purposes including,but not limited to, addition of one or more labels or binding moieties,alteration of mass, addition of specific sequences, or for any otherpurpose that would facilitate analysis of either the cleavage productsor analysis of any other by-product, result or consequence of thecleavage reaction.

[0278] Analysis of the cleavage products may involve subsequent steps orreactions that do not modify the cleavage products themselves. Forexample, cleavage products may be used to complete a functionalstructure, such as a competent promoter for in vitro transcription oranother protein binding site. Analysis may include the step of using thecompleted structure for or to perform its function. One or more cleavageproducts may also be used to complete an overlapping cleavage structure,thereby enabling a subsequent cleavage reaction, the products of whichmay be detected or used by any of the methods described herein,including the participation in further cleavage reactions.

[0279] Certain preferred embodiments of the invasive cleavage reactionsare provided in the following descriptions. In some embodiments, themethods of the present invention employ at least a pair ofoligonucleotides that interact with a target nucleic acid to form acleavage structure for a structure-specific nuclease. In someembodiments, the cleavage structure comprises i) a target nucleic acidthat may be either single-stranded or double-stranded (when adouble-stranded target nucleic acid is employed, it may be renderedsingle stranded, e.g., by heating); ii) a first oligonucleotide, termedthe “probe,” that defines a first region of the target nucleic acidsequence by being the complement of that region; iii) a secondoligonucleotide, termed the “INVADER oligonucleotide,” the 5′ part ofwhich defines a second region of the same target nucleic acid sequence,adjacent to and downstream of the first target region, and the secondpart of which overlaps into the region defined by the firstoligonucleotide.

[0280] It can be considered that the binding of these oligonucleotidesin this embodiment divides the target nucleic acid into three distinctregions: one region that has complementarity to only the probe; oneregion that has complementarity only to the INVADER oligonucleotide; andone region that has complementarity to both oligonucleotides. Asdiscussed above, in some preferred embodiments of the present invention,the overlap may comprise moieties other than overlapping complementarybases. Thus, in some embodiments, there is a physical, but not sequence,overlap between the INVADER and probe oligonucleotides, i.e., in theselatter embodiments, there is not a region of the target nucleic acidthat has complementarity to both oligonucleotides.

[0281] a) Oligonucleotide Design

[0282] Design of these oligonucleotides (i.e., the INVADERoligonucleotide and the probe) is accomplished using practices that arestandard in the art. For example, sequences that haveself-complementarity, such that the resulting oligonucleotides wouldeither fold upon themselves, or hybridize to each other at the expenseof binding to the target nucleic acid, are generally avoided.

[0283] One consideration in choosing a length for these oligonucleotidesis the complexity of the sample containing the target nucleic acid. Forexample, the human genome is approximately 3×10⁹ basepairs in length.Any 10-nucleotide sequence will appear with a frequency of 1:4¹⁰, or1:1,048,576 in a random string of nucleotides, which would beapproximately 2,861 times in 3 billion basepairs. Clearly, anoligonucleotide of this length would have a poor chance of bindinguniquely to a 10-nucleotide region within a target having a sequence thesize of the human genome. If the target sequence were within a 3 kbplasmid, however, such an oligonucleotide might have a very reasonablechance of binding uniquely. By this same calculation it can be seen thatan oligonucleotide of 16 nucleotides (i.e., a 16-mer) is the minimumlength of a sequence that is mathematically likely to appear once in3×10⁹ basepairs. This level of specificity may also be provided by twoor more shorter oligonucleotides if they are configured to bind in acooperative fashion (i.e., such that they can produce the intendedcomplex only if both or all are bound to their intended targetsequences), wherein the combination of the short oligonucleotidesprovides the desired specificity. In one such embodiment, thecooperativity between the shorter oligonucleotides is by a coaxialstacking effect that can occur when the oligonucleotides hybridize toadjacent sites on a target nucleic acid. In another embodiment, theshorter oligonucleotides are connected to one another, either directly,or by one or more spacer regions. The short oligonucleotides thusconnected may bind to distal regions of the target and may be used tobridge across regions of secondary structure in a target. Examples ofsuch bridging oligonucleotides are described in PCT Publication WO98/50403, herein incorporated by reference in its entirety.

[0284] A second consideration in choosing oligonucleotide length is thetemperature range in which the oligonucleotides will be expected tofunction. A 16-mer of average base content (50% G-C bases) will have acalculated T_(m) of about 41° C., depending on, among other things, theconcentration of the oligonucleotide and its target, the salt content ofthe reaction and the precise order of the nucleotides. As a practicalmatter, longer oligonucleotides are usually chosen to enhance thespecificity of hybridization. Oligonucleotides 20 to 25 nucleotides inlength are often used, as they are highly likely to be specific if usedin reactions conducted at temperatures that are near their T_(m)s(within about 5° C. of the T_(m)). In addition, with calculated T_(m)sin the range of 50 to 70° C., such oligonucleotides (i.e., 20 to25-mers) are appropriately used in reactions catalyzed by thermostableenzymes, which often display optimal activity near this temperaturerange.

[0285] The maximum length of the oligonucleotide chosen is also based onthe desired specificity. One should avoid choosing sequences that are solong that they are either at a high risk of binding stably to partialcomplements, or that they cannot easily be dislodged when desired (e.g.,failure to disassociate from the target once cleavage has occurred orfailure to disassociate at a reaction temperature suitable for theenzymes and other materials in the reaction).

[0286] The first step of design and selection of the oligonucleotidesfor the INVADER oligonucleotide-directed cleavage is in accordance withthese sample general principles. Considered as sequence-specific probesindividually, each oligonucleotide may be selected according to theguidelines listed above. That is to say, each oligonucleotide willgenerally be long enough to be reasonably expected to hybridize only tothe intended target sequence within a complex sample, usually in the 20to 40 nucleotide range. Alternatively, because the INVADERoligonucleotide-directed cleavage assay depends upon the concertedaction of these oligonucleotides, the composite length of the 2oligonucleotides which span/bind to the target may be selected to fallwithin this range, with each of the individual oligonucleotides being inapproximately the 13 to 17 nucleotide range. Such a design might beemployed if a non-thermostable cleavage means were employed in thereaction, requiring the reactions to be conducted at a lower temperaturethan that used when thermostable cleavage means are employed. In someembodiments, it may be desirable to have these oligonucleotides bindmultiple times within a single target nucleic acid (e.g., to bind tomultiple variants or multiple similar sequences within a target). It isnot intended that the method of the present invention be limited to anyparticular size of the probe or INVADER oligonucleotide.

[0287] The second step of designing an oligonucleotide pair for thisassay is to choose the degree to which the upstream “INVADER”oligonucleotide sequence will overlap into the downstream “probe”oligonucleotide sequence, and consequently, the sizes into which theprobe will be cleaved. In some preferred embodiments, the probeoligonucleotide can be made to “turn over,” that is to say probe can bemade to depart to allow the binding and cleavage of other copies of theprobe molecule, without the requirements of thermal denaturation ordisplacement by polymerization. While in one embodiment of this assayprobe turnover may be facilitated by an exonucleolytic digestion by thecleavage agent, in some preferred embodiments of the present inventionturnover does not require this exonucleolytic activity. For example, insome embodiments, a reaction temperature and reaction conditions areselected so as to create an equilibrium wherein the probe hybridizes anddisassociates from the target. In other embodiments, temperature andreaction conditions are selected so that unbound probe can initiatebinding to the target strand and physically displace bound probe. Instill other embodiments, temperature and reaction conditions areselected such that either or both mechanisms of probe replacement mayoccur in any proportion. The method of the present invention is notlimited to any particular mechanism of probe replacement. By anymechanism, when the probe is bound to the target to form a cleavagestructure, cleavage can occur. The continuous cycling of the probe onand off of the target allows multiple probes to bind and be cleaved foreach copy of a target nucleic acid.

[0288] i) Non-Sequence Overlaps

[0289] It has been determined that the relationship between the 3′ endof the upstream oligonucleotide and the desired site of cleavage on theprobe should be carefully designed. It is known that the preferred siteof cleavage for the types of structure-specific endonucleases employedherein is one basepair into a duplex (Lyamichev et al., supra). It waspreviously believed that the presence of an upstream oligonucleotide orprimer allowed the cleavage site to be shifted away from this preferredsite, into the single stranded region of the 5′ arm (Lyamichev et al.,supra and U.S. Pat. No. 5,422,253). In contrast to this previouslyproposed mechanism, and while not limiting the present invention to anyparticular mechanism, it is believed that the nucleotide immediately 5′,or upstream of the cleavage site on the probe (including miniprobe andmid-range probes) should be able to basepair with the target forefficient cleavage to occur. In the case of the present invention, thiswould be the nucleotide in the probe sequence immediately upstream ofthe intended cleavage site. In addition, as described herein, it hasbeen observed that in order to direct cleavage to that same site in theprobe, the upstream oligonucleotide should have its 3′ base (i.e., nt)immediately upstream of the intended cleavage site of the probe. Inembodiments where the INVADER and probe oligonucleotides share asequence overlap, this places the 3′ terminal nucleotide of the upstreamoligonucleotide and the base of the probe oligonucleotide 5′ of thecleavage site in competition for pairing with the correspondingnucleotide of the target strand.

[0290] To examine the outcome of this competition (i.e. which base ispaired during a successful cleavage event), substitutions were made inthe probe and INVADER oligonucleotides such that either the probe or theINVADER oligonucleotide were mismatched with the target sequence at thisposition. The effects of both arrangements on the rates of cleavage wereexamined. When the INVADER oligonucleotide is unpaired at the 3′ end,the rate of cleavage was not reduced. If this base was removed, however,the cleavage site was shifted upstream of the intended site. Incontrast, if the probe oligonucleotide was not base-paired to the targetjust upstream of the site to which the INVADER oligonucleotide wasdirecting cleavage, the rate of cleavage was dramatically reduced,suggesting that when a competition exists, the probe oligonucleotide wasthe molecule to be base-paired in this position.

[0291] It appears that the 3′ end of the upstream INVADERoligonucleotide is unpaired during cleavage, and yet is important foraccurate positioning of the cleavage. To examine which part(s) of the 3′terminal nucleotide are required for the positioning of cleavage,INVADER oligonucleotides were designed that terminated on this end withnucleotides that were altered in a variety of ways. Sugars examinedincluded 2′ deoxyribose with a 3′ phosphate group, a dideoxyribose, 3′deoxyribose, 2′ O-methyl ribose, arabinose and arabinose with a 3′phosphate. Abasic ribose, with and without 3′ phosphate were tested.Synthetic “universal” bases such at 3-nitropyrrole and 5-3 nitroindoleon ribose sugars were tested. Finally, a base-like aromatic ringstructure, acridine, linked to the 3′ end the previous nucleotidewithout a sugar group was tested. The results obtained support theconclusion that the aromatic ring of the base (at the 3′ end of theINVADER oligonuceotide) is an important moiety for accomplishing thedirection of cleavage to the desired site within the downstream probe.The 3′ terminal moiety of the INVADER oligonucleotide need not be a basethat is complementary to the target nucleic acid.

[0292] ii) Miniprobes and Mid-Range Probes;

[0293] As discussed above, the INVADER oligonucleotide-directed cleavageassay may be performed using INVADER and probe oligonucleotides thathave a length of about 13-25 nucleotides (typically 20-25 nucleotides).It is also contemplated that the oligonucleotides may themselves becomposed of shorter oligonucleotide sequences that align along a targetstrand but that are not covalently linked. This is to say that there isa nick in the sugar-phosphate backbone of the composite oligonucleotide,but that there is no disruption in the progression of base-pairednucleotides in the resulting duplex. When short strands of nucleic acidalign contiguously along a longer strand the hybridization of each isstabilized by the hybridization of the neighboring fragments because thebasepairs can stack along the helix as though the backbone was in factuninterrupted. This cooperativity of binding can give each segment astability of interaction in excess of what would be expected for thesegment hybridizing to the longer nucleic acid alone. One application ofthis observation has been to assemble primers for DNA sequencing,typically about 18 nucleotides long, from sets of three hexameroligonucleotides that are designed to hybridize in this way (Kotler etal. Proc. Natl. Acad. Sci. USA 90:4241 [1993]). The resultingdoubly-nicked primer can be extended enzymatically in reactionsperformed at temperatures that might be expected to disrupt thehybridization of hexamers, but not of 18-mers.

[0294] The use of composite or split oligonucleotides is applied withsuccess in the INVADER-directed cleavage assay. For example, the probeoligonucleotide may be split into two oligonucleotides that anneal in acontiguous and adjacent manner along a target oligonucleotide such thatthe downstream oligonucleotide (analogous to the probe) is assembledfrom two smaller pieces: a short segment of 6-10 nts (termed the“miniprobe”), that is to be cleaved in the course of the detectionreaction, and an oligonucleotide that hybridizes immediately downstreamof the miniprobe (termed the “stacker”), that serves to stabilize thehybridization of the probe. To form the cleavage structure, an upstreamoligonucleotide (the INVADER oligonucleotide) is provided to direct thecleavage activity to the desired region of the miniprobe. Assembly ofthe probe from non-linked pieces of nucleic acid (i.e., the miniprobeand the stacker) allows regions of sequences to be changed withoutrequiring the re-synthesis of the entire proven sequence, thus improvingthe cost and flexibility of the detection system. In addition, the useof unlinked composite oligonucleotides makes the system more stringentin its requirement of perfectly matched hybridization to achieve signalgeneration, allowing this to be used as a sensitive means of detectingmutations or changes in the target nucleic acid sequences.

[0295] In one embodiment, the methods of the present invention employ atleast three oligonucleotides that interact with a target nucleic acid toform a cleavage structure for a structure-specific nuclease. Morespecifically, the cleavage structure comprises i) a target nucleic acidthat may be either single-stranded or double-stranded (when adouble-stranded target nucleic acid is employed, it may be renderedsingle-stranded, e.g., by heating); ii) a first oligonucleotide, termedthe “stacker,” that defines a first region of the target nucleic acidsequence by being the complement of that region; iii) a secondoligonucleotide, termed the “miniprobe,” that defines a second region ofthe target nucleic acid sequence by being the complement of that region;iv) a third oligonucleotide, termed the “INVADER oligonucleotide,” the5′ part of which defines a third region of the same target nucleic acidsequence, adjacent to and downstream of the second target region, andthe second or 3′ part of which overlaps into the region defined by thesecond oligonucleotide As described above for embodiments that do notemploy a stacker, the overlap region can represent a region where thereis a physical, but not sequence, overlap between the INVADER and probeoligonucleotides.

[0296] In addition to the benefits cited above, the use of a compositedesign for the oligonucleotides that form the cleavage structure allowsmore latitude in the design of the reaction conditions for performingthe INVADER-directed cleavage assay. When a longer probe (e.g., 16-25nt), as described above, is used for detection in reactions that areperformed at temperatures below the T_(m) of that probe, the cleavage ofthe probe may play a significant role in destabilizing the duplex ofwhich it is a part, thus allowing turnover and reuse of the recognitionsite on the target nucleic acid. In contrast, reaction temperatures thatare at or above the T_(m) of the probe mean that the probe molecules arehybridizing and releasing from the target quite rapidly even withoutcleavage of the probe. When an upstream INVADER oligonucleotide and acleavage agent are provided the probe will be specifically cleaved, butthe cleavage will not be necessary to the turnover of the probe. When along probe (e.g., 16-25 nt) is used in this way the temperaturesrequired to achieve this state is high, around 65 to 70° C. for a 25-merof average base composition. Requiring the use of such elevatedtemperatures limits the choice of cleavage agents to those that are verythermostable, and may contribute to background in the reactions,depending of the means of detection, through thermal degradation of theprobe oligonucleotides. With miniprobes, this latter mechanism of probereplacement may be accomplished at a lower temperature. Thus, shorterprobes are preferred for embodiments using lower reaction temperatures.

[0297] The miniprobe of the present invention may vary in size dependingon the desired application. In one embodiment, the probe may berelatively short compared to a standard probe (e.g., 16-25 nt), in therange of 6 to 10 nucleotides. When such a short probe is used, reactionconditions can be chosen that prevent hybridization of the miniprobe inthe absence of the stacker oligonucleotide. In this way a short probecan be made to assume the statistical specificity and selectivity of alonger sequence. In the event of a perturbation in the cooperativebinding of the miniprobe and stacker nucleic acids, as might be causedby a mismatch within the short sequence that is otherwise complementaryto the target nucleic acid or at the junction between the contiguousduplexes, this cooperativity can be lost, dramatically reducing thestability of the shorter duplex (i.e., that of the miniprobe), and thusreducing the level of cleaved product in the assay of the presentinvention.

[0298] It is also contemplated that probes of intermediate size may beused. Such probes, in the 11 to 15 nucleotide range, may blend some ofthe features associated with the longer probes as originally described,these features including the ability to hybridize and be cleaved absentthe help of a stacker oligonucleotide. At temperatures below theexpected T_(m) of such probes, the mechanisms of turnover may be asdiscussed above for probes in the 20 nt range, and be dependent on theremoval of the sequence in the overlap region for destabilization andcycling.

[0299] The mid-range probes may also be used at elevated temperatures,at or above their expected T_(m), to allow melting rather than cleavageto promote probe turnover. In contrast to the longer probes describedabove, however, the temperatures required to allow the use of such athermally driven turnover are much lower (about 40 to 60° C.), thuspreserving both the cleavage means and the nucleic acids in the reactionfrom thermal degradation. In this way, the mid-range probes may performin some instances like the miniprobes described above. In a furthersimilarity to the miniprobes, the accumulation of cleavage signal from amid-range probe may be helped under some reaction conditions by thepresence of a stacker.

[0300] To summarize, a standard long probe usually does not benefit fromthe presence of a stacker oligonucleotide downstream (the exceptionbeing cases where such an oligonucleotide may also disrupt structures inthe target nucleic acid that interfere with the probe binding), and itmay be used in conditions requiring several nucleotides to be removed toallow the oligonucleotide to release from the target efficiently. Iftemperature of the reaction is used to drive exchange of the probes,standard probes may require use of a temperature at which nucleic acidsand enzymes are at higher risk of thermal degradation.

[0301] The miniprobe is very short and performs optimally in thepresence of a downstream stacker oligonucleotide. The miniprobes arewell suited to reactions conditions that use the temperature of thereaction to drive rapid exchange of the probes on the target regardlessof whether any bases have been cleaved. In reactions with sufficientamount of the cleavage means, the probes that do bind will be rapidlycleaved before they melt off.

[0302] The mid-range or midiprobe combines features of these probes andcan be used in reactions like those favored by long probes, with longerregions of overlapto drive probe turnover at lower temperature. In apreferred embodiment, the midrange probes are used at temperaturessufficiently high that the probes are hybridizing to the target andreleasing rapidly regardless of cleavage. The mid-range probe may haveenhanced performance in the presence of a stacker under somecircumstances.

[0303] The distinctions between the mini-, midi-(i.e., mid-range) andlong probes are not contemplated to be inflexible and based only onlength. The performance of any given probe may vary with its specificsequence, the choice of solution conditions, the choice of temperatureand the selected cleavage means.

[0304] The assemblage of oligonucleotides that comprises the cleavagestructure of the present invention is sensitive to mismatches betweenthe probe and the target. It is also contemplated that a mismatchbetween the INVADER oligonucleotide and the target may be used todistinguish related target sequences. In the 3-oligonucleotide system,comprising an INVADER, a probe and a stacker oligonucleotide, it iscontemplated that mismatches may be located within any of the regions ofduplex formed between these oligonucleotides and the target sequence. Ina preferred embodiment, a mismatch to be detected is located in theprobe. In a particularly preferred embodiment, the mismatch is in theprobe, at the basepair immediately upstream (i.e., 5′) of the site thatis cleaved when the probe is not mismatched to the target.

[0305] In another preferred embodiment, a mismatch to be detected islocated within the region defined by the hybridization of a miniprobe.In a particularly preferred embodiment, the mismatch is in theminiprobe, at the basepair immediately upstream (i.e., 5′) of the sitethat is cleaved when the miniprobe is not mismatched to the target.

[0306] iii) Software for Oligonucleotide Design for the INVADER Assay

[0307] The present invention provides systems and methods for the designof oligonucleotides for use in detection assays. In particular, thepresent invention provides systems and methods for the design ofoligonucleotides that successfully hybridize to appropriate regions oftarget nucleic acids (e.g., regions of target nucleic acids that do notcontain secondary structure) under the desired reaction conditions(e.g., temperature, buffer conditions, etc.) for the detection assay.The systems and methods also allow for the design of multiple differentoligonucleotides (e.g., oligonucleotides that hybridize to differentportions of a target nucleic acid or that hybridize to two or moredifferent target nucleic acids) that all function in the detection assayunder the same or substantially the same reaction conditions. Thesesystems and methods may also be used to design control samples that workunder the experimental reaction conditions.

[0308] While the systems and methods of the present invention are notlimited to any particular detection assay, the following descriptionillustrates the invention when used in conjunction with the INVADERassay (Third Wave Technologies, Madison Wis.; See e.g., U.S. Pat. Nos.5,846,717, 5,985,557, 5,994,069, and 6,001,567 and PCT Publications WO97/27214 and WO 98/42873, incorporated herein by reference in theirentireties) to detect a SNP. One skilled in the art will appreciate thatspecific and general features of this illustrative example are generallyapplicable to other detection assays, and for use in designing INVADERassays for purposes other than SNP detection (e.g., for DNA or RNAquantitation, for RNA splice junction detection, etc.). Further, it willbe appreciated that all algorithms described herein can be applied asseparate software elements, or calculations may be performed manually,for the design of any INVADER assay probe set without use of theINVADERCREATOR design system described below.

[0309] Oligonucleotide Design for the INVADER Assay Using theINVADERCREATOR Program

[0310] In some embodiments where an oligonucleotide is designed for usein the INVADER assay to detect a SNP, the sequence(s) of interest areentered into the INVADERCREATOR program (Third Wave Technologies,Madison, Wis.). As described above, sequences may be input for analysisfrom any number of sources, either directly into the computer hostingthe INVADERCREATOR program, or via a remote computer linked through acommunication network (e.g., a LAN, Intranet or Internet network). Theprogram designs probes for both the sense and antisense strand. Strandselection is generally based upon the ease of synthesis, minimization ofsecondary structure formation, and manufacturability. In someembodiments, the user chooses the strand for sequences to be designedfor. In other embodiments, the software automatically selects thestrand. By incorporating thermodynamic parameters for optimum probecycling and signal generation (Allawi and SantaLucia, Biochemistry,36:10581 [1997]), oligonucleotide probes may be designed to operate at apre-selected assay temperature (e.g., 63° C.). Based on these criteria,a final probe set (e.g., primary probes for 2 alleles and an INVADERoligonucleotide) is selected.

[0311] In some embodiments, the INVADERCREATOR system is a web-basedprogram with secure site access that contains a link to BLAST (availableat the National Center for Biotechnology Information, National Libraryof Medicine, National Institutes of Health web site) and that can belinked to RNAstructure (Mathews et al., RNA 5:1458 [1999]), a softwareprogram that incorporates mfold (Zuker, Science, 244:48 [1989]).RNAstructure tests the proposed oligonucleotide designs generated byINVADERCREATOR for potential uni- and bimolecular complex formation.INVADERCREATOR is open database connectivity (ODBC)-compliant and usesthe Oracle database for export/integration. The INVADERCREATOR systemwas configured with Oracle to work well with UNIX systems, as mostgenome centers are UNIX-based.

[0312] In some embodiments, the INVADERCREATOR analysis is provided on aseparate server (e.g., a Sun server) so it can handle analysis of largebatch jobs. For example, a customer can submit up to 2,000 SNP sequencesin one email. The server passes the batch of sequences on to theINVADERCREATOR software, and, when initiated, the program designs SNPsets. In some embodiments, probe set designs are returned to the userwithin 24 hours of receipt of the sequences.

[0313] In some preferred embodiments, each INVADER assay reactionincludes at least two target sequence-specific, unlabeledoligonucleotides for the primary reaction: an upstream INVADERoligonucleotide and a downstream Probe oligonucleotide. The INVADERoligonucleotide is generally designed to bind stably at the reactiontemperature, while the probe is designed to freely associate anddisassociate with the target strand, with cleavage occurring only whenan uncut probe hybridizes adjacent to an overlapping INVADERoligonucleotide. In some embodiments, the probe includes a 5′ flap or“arm” that is not complementary to the target, and this flap is releasedfrom the probe when cleavage occurs. In some embodiments, the releasedflap participates as an INVADER oligonucleotide in a secondary reaction.

[0314] The following discussion provides one example of how a userinterface for an INVADERCREATOR program may be configured.

[0315] The user opens a work screen (FIG. 42), e.g., by clicking on anicon on a desktop display of a computer (e.g., a Windows desktop). Theuser enters information related to the target sequence for which anassay is to be designed. In some embodiments, the user enters a targetsequence. In other embodiments, the user enters a code or number thatcauses retrieval of a sequence from a database. In still otherembodiments, additional information may be provided, such as the user'sname, an identifying number associated with a target sequence, and/or anorder number. In preferred embodiments, the user indicates (e.g. via acheck box or drop down menu) that the target nucleic acid is DNA or RNA.In other preferred embodiments, the user indicates the species fromwhich the nucleic acid is derived. In particularly preferredembodiments, the user indicates whether the design is for monoplex(i.e., one target sequence or allele per reaction) or multiplex (i.e.,multiple target sequences or alleles per reaction) detection. When therequisite choices and entries are complete, the user starts the analysisprocess. In one embodiment, the user clicks a “Go Design It” button tocontinue.

[0316] In some embodiments, the software validates the field entriesbefore proceeding. In some embodiments, the software verifies that anyrequired fields are completed with the appropriate type of information.In other embodiments, the software verifies that the input sequencemeets selected requirements (e.g., minimum or maximum length, DNA or RNAcontent). If entries in any field are not found to be valid, an errormessage or dialog box may appear. In preferred embodiments, the errormessage indicates which field is incomplete and/or incorrect. Once asequence entry is verified, the software proceeds with the assay design.

[0317] In some embodiments, the information supplied in the order entryfields specifies what type of design will be created. In preferredembodiments, the target sequence and multiplex check box specify whichtype of design to create. Design options include but are not limited toSNP assay, Multiplexed SNP assay (e.g., wherein probe sets for differentalleles are to be combined in a single reaction), Multiple SNP assay(e.g., wherein an input sequence has multiple sites of variation forwhich probe sets are to be designed), and Multiple Probe Arm assays.

[0318] In some embodiments, the INVADERCREATOR software is started via aWeb Order Entry (WebOE) process (i.e., through an Intra/Internet browserinterface) and these parameters are transferred from the WebOE viaapplet <param> tags, rather than entered through menus or check boxes.

[0319] In the case of Multiple SNP Designs, the user chooses two or moredesigns to work with. In some embodiments, this selection opens a newscreen view (e.g., a Multiple SNP Design Selection view FIG. 43). Insome embodiments, the software creates designs for each locus in thetarget sequence, scoring each, and presents them to the user in thisscreen view. The user can then choose any two designs to work with. Insome embodiments, the user chooses a first and second design (e.g., viaa menu or buttons) and clicks a “Go Design It” button to continue.

[0320] To select a probe sequence that will perform optimally at apre-selected reaction temperature, the melting temperature (T_(m)) ofthe SNP to be detected is calculated using the nearest-neighbor modeland published parameters for DNA duplex formation (Allawi andSantaLucia, Biochemistry, 36:10581 [1997]). In embodiments wherein thetarget strand is RNA, parameters appropriate for RNA/DNA heteroduplexformation may be used. Because the assay's salt concentrations are oftendifferent than the solution conditions in which the nearest-neighborparameters were obtained (1M NaCl and no divalent metals), and becausethe presence and concentration of the enzyme influence optimal reactiontemperature, an adjustment should be made to the calculated T_(m) todetermine the optimal temperature at which to perform a reaction. Oneway of compensating for these factors is to vary the value provided forthe salt concentration within the melting temperature calculations. Thisadjustment is termed a ‘salt correction’. As used herein, the term “saltcorrection” refers to a variation made in the value provided for a saltconcentration for the purpose of reflecting the effect on a T_(m)calculation for a nucleic acid duplex of a non-salt parameter orcondition affecting said duplex. Variation of the values provided forthe strand concentrations will also affect the outcome of thesecalculations. By using a value of 0.5 M NaCl (SantaLucia, Proc Natl AcadSci USA, 95:1460 [1998]) and strand concentrations of about 1 mM of theprobe and 1 fM target, the algorithm used for calculating probe-targetmelting temperature has been adapted for use in predicting optimalINVADER assay reaction temperature. For a set of 30 probes, the averagedeviation between optimal assay temperatures calculated by this methodand those experimentally determined is about 1.5° C.

[0321] The length of the downstream probe analyte-specific region (ASR)is defined by the temperature selected for running the reaction (e.g.,63° C.). Starting from the position of the variant nucleotide on thetarget DNA (the target base that is paired to the probe nucleotide 5′ ofthe intended cleavage site), and adding on the 3′ end, an iterativeprocedure is used by which the length of the target-binding region ofthe probe is increased by one base pair at a time until a calculatedoptimal reaction temperature (T_(m) plus salt correction to compensatefor enzyme effect) matching the desired reaction temperature is reached.The non-complementary arm of the probe is preferably selected to allowthe secondary reaction to cycle at the same reaction temperature. Theentire probe oligonucleotide is screened using programs such as mfold(Zuker, Science, 244: 48 [1989]) or Oligo 5.0 (Rychlik and Rhoads,Nucleic Acids Res, 17: 8543 [1989]) for the possible formation of dimercomplexes or secondary structures that could interfere with thereaction. The same principles are also followed for INVADERoligonucleotide design. Briefly, starting from the position N on thetarget DNA, the 3′ end of the INVADER oligonucleotide is designed tohave a nucleotide not complementary to either allele suspected of beingcontained in the sample to be tested. The mismatch does not adverselyaffect cleavage (Lyamichev et al., Nature Biotechnology, 17: 292[1999]), and it can enhance probe cycling, presumably by minimizingcoaxial stabilization effects between the two probes. Additionalresidues complementary to the target DNA starting from residue N−1 arethen added in the 5′ direction until the stability of theINVADERoligonucleotide-target hybrid exceeds that of the probe (andtherefore the planned assay reaction temperature), generally by 15-20°C.

[0322] In some embodiments, the released cleavage fragment from aprimary reaction is to be used in a secondary reaction. It is one aspectof the assay design that the all of the probe sequences may be selectedto allow the primary and secondary reactions to occur at the sameoptimal temperature, so that the reaction steps can run simultaneously.In an alternative embodiment, the probes may be designed to operate atdifferent optimal temperatures, so that the reaction steps are notsimultaneously at their temperature optima.

[0323] In some embodiments, the software provides the user anopportunity to change various aspects of the design including but notlimited to: probe, target and INVADER oligonucleotide temperature optimaand concentrations; blocking groups; probe arms; dyes, capping groupsand other adducts; individual bases of the probes and targets (e.g.,adding or deleting bases from the end of targets and/or probes, orchanging internal bases in the INVADER and/or probe and/or targetoligonucleotides). In some embodiments, changes are made by selectionfrom a menu. In other embodiments, changes are entered into text ordialog boxes. In preferred embodiments, this option opens a new screen(e.g., a Designer Worksheet view, FIG. 44).

[0324] In some embodiments, the software provides a scoring system toindicate the quality (e.g., the likelihood of performance) of the assaydesigns. In one embodiment, the scoring system includes a starting scoreof points (e.g., 100 points) wherein the starting score is indicative ofan ideal design, and wherein design features known or suspected to havean adverse affect on assay performance are assigned penalty values.Penalty values may vary depending on assay parameters other than thesequences, including but not limited to the type of assay for which thedesign is intended (e.g., monoplex, multiplex) and the temperature atwhich the assay reaction will be performed. The following exampleprovides illustrative scoring criteria for use with some embodiments ofthe INVADER assay based on an intelligence defined by experimentation.Examples of design features that may incur score penalties include butare not limited to the following [penalty values are indicated inbrackets, first number is for lower temperature assays (e.g., 62-64°C.), second is for higher temperature assays (e.g., 65-66° C.)]:

[0325] 1. [100:100] 3′ end of INVADER oligonucleotide resembles theprobe arm:

[0326] ARM SEQUENCE: PENALTY AWARDED IF INVADER

[0327] OLIGONUCLEOTIDE ENDS IN: Arm 1: CGCGCCGAGG 5′ . . . GAGGX or 5′ .. . GAGGXX Arm 2: ATGACGTGGCAGAC 5′ . . . CAGACX or 5′ . . . CAGACXX Arm3: ACGGACGCGGAG 5′ . . . GGAGX or 5′ . . . GGAGXX Arm 4: TCCGCGCGTCC5′ . . . GTCCX or 5′ . . . GTCCXX

[0328] 2. [70:70] a probe has 5-base stretch (i.e., 5 of the same basein a row) containing the polymorphism;

[0329] 3. [60:60] a probe has 5-base stretch adjacent to thepolymorphism;

[0330] 4. [50:50] a probe has 5-base stretch one base from thepolymorphism;

[0331] 5. [40:40] a probe has 5-base stretch two bases from thepolymorphism;

[0332] 6. [50:50] probe 5-base stretch is of Gs—additional penalty;

[0333] 7. [100:100] a probe has 6-base stretch anywhere;

[0334] 8. [90:90] a two or three base sequence repeats at least fourtimes;

[0335] 9. [100:100] a degenerate base occurs in a probe;

[0336] 10. [60:90] probe hybridizing region is short (13 bases or lessfor designs 65-67° C.; 12 bases or less for designs 62-64° C.)

[0337] 11. [40:90] probe hybridizing region is long (29 bases or morefor designs 65-67° C., 28 bases or more for designs 62-64° C.)

[0338] 12. [5:5] probe hybridizing region length—per base additionalpenalty

[0339] 13. [80:80] Ins/Del design with poor discrimination in first 3bases after probe arm

[0340] 14. [100:100] calculated INVADER oligonucleotide T_(m) within7.5° C. of probe target T_(m) (designs 65-67° C. with INVADERoligonucleotide less than ≦70.5° C., designs 62-64° C. with INVADERoligonucleotide ≦69.5° C.

[0341] 15. [20:20] calculated probes T_(m)s differ by more than 2.0° C.

[0342] 16. [100:100] a probe has calculated T_(m) 2° C. less than itstarget T_(m)

[0343] 17. [10:10] target of one strand 8 bases longer than that ofother strand

[0344] 18. [30:30] INVADER oligonucleotide has 6-base stretchanywhere—initial penalty

[0345] 19. [70:70] INVADER oligonucleotide 6-base stretch is ofGs—additional penalty

[0346] 20. [15:15] probe hybridizing region is 14, 15 or 24-28 baseslong (65-67° C.) or 13,14 or 26,27 bases long (62-64° C.)

[0347] 21. [15:15] a probe has a 4-base stretch of Gs containing thepolymorphism

[0348] In particularly preferred embodiments, temperatures for each ofthe oligonucleotides in the designs are recomputed and scores arerecomputed as changes are made. In some embodiments, score descriptionscan be seen by clicking a “descriptions” button. In some embodiments, aBLAST search option is provided. In preferred embodiments, a BLASTsearch is done by clicking a “BLAST Design” button. In some embodiments,this action brings up a dialog box describing the BLAST process. Inpreferred embodiments, the BLAST search results are displayed as ahighlighted design on a Designer Worksheet.

[0349] In some embodiments, a user accepts a design by clicking an“Accept” button. In other embodiments, the program approves a designwithout user intervention. In preferred embodiments, the program sendsthe approved design to a next process step (e.g., into production; intoa file or database). In some embodiments, the program provides a screenview (e.g., an Output Page, FIG. 45), allowing review of the finaldesigns created and allowing notes to be attached to the design. Inpreferred embodiments, the user can return to the Designer Worksheet(e.g., by clicking a “Go Back” button) or can save the design (e.g., byclicking a “Save It” button) and continue (e.g., to submit the designedoligonucleotides for production).

[0350] In some embodiments, the program provides an option to create ascreen view of a design optimized for printing (e.g., a text-only view)or other export (e.g., an Output view, FIG. 46). In preferredembodiments, the Output view provides a description of the designparticularly suitable for printing, or for exporting into anotherapplication (e.g., by copying and pasting into another application). Inparticularly preferred embodiments, the Output view opens in a separatewindow.

[0351] The present invention is not limited to the use of theINVADERCREATOR software. Indeed, a variety of software programs arecontemplated and are commercially available, including, but not limitedto GCG Wisconsin Package (Genetics computer Group, Madison, Wis.) andVector NTI (Informax, Rockville, Md.).

[0352] b) Design of the Reaction Conditions

[0353] Target nucleic acids (e.g., RNA and DNA) that may be analyzedusing the methods of the present invention that employ a 5′ nuclease orother appropriate cleavage agents. Such nucleic acids may be obtainedusing standard molecular biological techniques. For example, nucleicacids (RNA or DNA) may be isolated from a tissue sample (e.g., a biopsyspecimen), tissue culture cells, samples containing bacteria and/orviruses (including cultures of bacteria and/or viruses), etc. The targetnucleic acid may also be transcribed in vitro from a DNA template or maybe chemically synthesized or amplified in by polymerase chain reaction.Furthermore, nucleic acids may be isolated from an organism, either asgenomic material or as a plasmid or similar extrachromosomal DNA, orthey may be a fragment of such material generated by treatment with arestriction endonuclease or other cleavage agent, or a shearing force,or it may be synthetic.

[0354] Assembly of the target, probe, and INVADER oligonucleotidenucleic acids into the cleavage reaction of the present invention usesprinciples commonly used in the design of oligonucleotide-basedenzymatic assays, such as dideoxynucleotide sequencing and polymerasechain reaction (PCR). As is done in these assays, the oligonucleotidesare provided in sufficient excess that the rate of hybridization to thetarget nucleic acid is very rapid. These assays are commonly performedwith 50 fmoles to 2 pmoles of each oligonucleotide per microliter ofreaction mixture, although they are not necessarily limited to thisrange. In the Examples described herein, amounts of oligonucleotidesranging from 250 fmoles to 5 pmoles per microliter of reaction volumewere used. These values were chosen for the purpose of ease indemonstration and are not intended to limit the performance of thepresent invention to these concentrations. Other (e.g., lower)oligonucleotide concentrations commonly used in other molecularbiological reactions are also contemplated.

[0355] It is desirable that an INVADER oligonucleotide be immediatelyavailable to direct the cleavage of each probe oligonucleotide thathybridizes to a target nucleic acid. In some embodiments describedherein, the INVADER oligonucleotide is provided in excess over the probeoligonucleotide. While this is an effective means of making the INVADERoligonucleotide immediately available in such embodiments it is notintended that the practice of the present invention be limited toconditions wherein the INVADER oligonucleotide is in excess over theprobe, or to any particular ratio of INVADER-to-probe (e.g., in somepreferred embodiments described herein, the probe is provided in excessover the INVADER oligonucleotide). Another means of assuring thepresence of an INVADER oligonucleotide whenever a probe binds to atarget nucleic acid is to design the INVADER oligonucleotide tohybridize more stably to the target, i.e., to have a higher T_(m) thanthe probe. This can be accomplished by any of the means of increasingnucleic acid duplex stability discussed herein (e.g. by increasing theamount of complementarity to the target nucleic acid).

[0356] Buffer conditions should be chosen that are compatible with boththe oligonucleotide/target hybridization and with the activity of thecleavage agent. The optimal buffer conditions for nucleic acidmodification enzymes, and particularly DNA modification enzymes,generally included enough mono- and di-valent salts to allow associationof nucleic acid strands by base-pairing. If the method of the presentinvention is performed using an enzymatic cleavage agent other thanthose specifically described here, the reactions may generally beperformed in any such buffer reported to be optimal for the nucleasefunction of the cleavage agent. In general, to test the utility of anycleavage agent in this method, test reactions are performed wherein thecleavage agent of interest is tested in the MOPS/MnCl₂/KCl buffer orMg-containing buffers described herein and in whatever buffer has beenreported to be suitable for use with that agent, in a manufacturer'sdata sheet, a journal article, or in personal communication.

[0357] The products of the INVADER oligonucleotide-directed cleavagereaction are fragments generated by structure-specific cleavage of theinput oligonucleotides. The resulting cleaved and/or uncleavedoligonucleotides may be analyzed and resolved by a number of methodsincluding, but not limited to, electrophoresis (on a variety of supportsincluding acrylamide or agarose gels, paper, etc.), chromatography,fluorescence polarization, mass spectrometry and chip hybridization. Insome Examples the invention is illustrated using electrophoreticseparation for the analysis of the products of the cleavage reactions.However, it is noted that the resolution of the cleavage products is notlimited to electrophoresis. Electrophoresis is chosen to illustrate themethod of the invention because electrophoresis is widely practiced inthe art and is easily accessible to the average practitioner. In otherExamples, the invention is illustrated without electrophoresis or anyother resolution of the cleavage products.

[0358] The probe and INVADER oligonucleotides may contain a label to aidin their detection following the cleavage reaction. The label may be aradioisotope (e.g., a ³²P or ³⁵S-labelled nucleotide) placed at eitherthe 5′ or 3′ end of the oligonucleotide or alternatively, the label maybe distributed throughout the oligonucleotide (i.e., a uniformly labeledoligonucleotide). The label may be a nonisotopic detectable moiety, suchas a fluorophore, that can be detected directly, or a reactive groupthat permits specific recognition by a secondary agent. For example,biotinylated oligonucleotides may be detected by probing with astreptavidin molecule that is coupled to an indicator (e.g., alkalinephosphatase or a fluorophore) or a hapten such as dioxigenin may bedetected using a specific antibody coupled to a similar indicator. Thereactive group may also be a specific configuration or sequence ofnucleotides that can bind or otherwise interact with a secondary agent,such as another nucleic acid, and enzyme, or an antibody. In someembodiments, a probe is labeled with fluorescing moiety and a quenchingmoiety, wherein cleavage of the cleavage structure separates thefluorescing moiety from the quenching moiety, resulting in a detectablesignal (e.g., FRET detection). In some embodiments, a change inquenching of signal from a donor fluorophor is detected, while in otherembodiments, a change in emission from an acceptor fluorophore isdetected. In still other embodiments, the effect of FRET on both donorand acceptor emissions are detected.

[0359] In some embodiments of FRET detection, the fluorescence lifetimeof the fluorescence emitter is measured (e.g., as in time-resolvedfluorescence). While not limiting time-resolved fluorescence detectionembodiments to any particular labeling systems, examples of tags thatare useful in time-resolved FRET measurements include europium chelate(Eu³⁺; Biosclair, et al, J. Biomolecular Screening 5(5):319 [2000]),europium trisbipyridine cryptate (TBPEu³⁺; Alpha-Bazin, et al., Anal.Biochem. 286(1):17 [2000]), and ruthenium ligand complex{[Ru(bpy)2(phen-ITC)]²⁺; Youn, et al, Anal. Biochem. 232(1):24 [1995];Lakowicz, et al., Anal. Biochem. 288:62 [2001]).

[0360] c) Optimization of Reaction Conditions

[0361] The INVADER oligonucleotide-directed cleavage reaction is usefulto detect the presence of specific nucleic acids. In addition to theconsiderations listed above for the selection and design of the INVADERand probe oligonucleotides, the conditions under which the reaction isto be performed may be optimized for detection of a specific targetsequence.

[0362] One objective in optimizing the INVADER oligonucleotide-directedcleavage assay is to allow specific detection of the fewest copies of atarget nucleic acid. To achieve this end, it is desirable that thecombined elements of the reaction interact with the maximum efficiency,so that the rate of the reaction (e.g., the number of cleavage eventsper minute) is maximized. Elements contributing to the overallefficiency of the reaction include the rate of hybridization, the rateof cleavage, and the efficiency of the release of the cleaved probe.

[0363] The rate of cleavage will be a function of the cleavage meanschosen, and may be made optimal according to the manufacturer'sinstructions when using commercial preparations of enzymes or asdescribed in the examples herein. The other elements (rate ofhybridization, efficiency of release) depend upon the execution of thereaction, and optimization of these elements is discussed below.

[0364] Three elements of the cleavage reaction that significantly affectthe rate of nucleic acid hybridization are the concentration of thenucleic acids, the temperature at which the cleavage reaction isperformed and the concentration of salts and/or other charge-shieldingions in the reaction solution.

[0365] The concentrations at which oligonucleotide probes are used inassays of this type are well known in the art, and are discussed above.One example of a common approach to optimizing an oligonucleotideconcentration is to choose a starting amount of oligonucleotide forpilot tests; 0.01 to 2 μM is a concentration range used in manyoligonucleotide-based assays. When initial cleavage reactions areperformed, the following questions may be asked of the data: Is thereaction performed in the absence of the target nucleic acidsubstantially free of the cleavage product?; Is the site of cleavagespecifically positioned in accordance with the design of the INVADERoligonucleotide?; Is the specific cleavage product easily detected inthe presence of the uncleaved probe (or is the amount of uncut materialoverwhelming the chosen visualization method)?

[0366] A negative answer to any of these questions would suggest thatthe probe concentration is too high, and that a set of reactions usingserial dilutions of the probe should be performed until the appropriateamount is identified. Once identified for a given target nucleic acid ina give sample type (e.g., purified genomic DNA, body fluid extract,lysed bacterial extract), it should not need to be re-optimized. Thesample type is important because the complexity of the material presentmay influence the probe concentration optimum.

[0367] Conversely, if the chosen initial probe concentration is too low,the reaction may be slow, due to inefficient hybridization. Tests withincreasing quantities of the probe will identify the point at which theconcentration exceeds the optimum (e.g., at which it produces anundesirable effect, such as background cleavage not dependent on thetarget sequence, or interference with detection of the cleavedproducts). Since the hybridization will be facilitated by excess ofprobe, it is desirable, but not required, that the reaction be performedusing probe concentrations just below this point.

[0368] The concentration of INVADER oligonucleotide can be chosen basedon the design considerations discussed above. In some embodiments, theINVADER oligonucleotide is in excess of the probe oligonucleotide. In apreferred embodiment, the probe oligonucleotide is in excess of theINVADER oligonucleotide.

[0369] Temperature is also an important factor in the hybridization ofoligonucleotides. The range of temperature tested will depend in largepart on the design of the oligonucleotides, as discussed above. Where itis desired to have a reaction be run at a particular temperature (e.g.,because of an enzyme requirement, for convenience, for compatibilitywith assay or detection apparatuses, etc.), the oligonucleotides thatfunction in the reaction can be designed to optimally perform at thedesired reaction temperature. Each INVADER reaction includes at leasttwo target sequence-specific oligonucleotides for the primary reaction:an upstream INVADER oligonucleotide and a downstream probeoligonucleotide. In some preferred embodiments, the INVADERoligonucleotide is designed to bind stabily at the reaction temperature,while the probe is designed to freely associate and disassociate withthe target strand, with cleavage occurring only when an uncut probehybridizes adjacent to an overlapping INVADER oligonucleotide. Inpreferred embodiments, the probe includes a 5′ flap that is notcomplementary to the target, and this flap is released from the probewhen cleavage occurs. The released flap can be detected directly orindirectly. In some preferred embodiments, as discussed in detail below,the released flap participate as in INVADER oligonucleotide in asecondary reaction.

[0370] Optimum conditions for the INVADER assay are generally those thatallow specific detection of the smallest amount of a target nucleicacid. Such conditions may be characterized as those that yield thehighest target-dependent signal in a given timeframe, or for a givenamount of target nucleic acid, or that allow the highest rate of probecleavage (i.e., probes cleaved per minute).

[0371] As noted above, the concentration of the cleavage agent canaffect the actual optimum temperature for a cleavage reaction.Additionally, different cleavage agents, even if used at identicalconcentrations, can affect reaction temperature optima differently(e.g., the difference between the calculated probe T_(m) and theobserved optimal reaction temperature may be greater for one enzyme thanfor another). Determination of appropriate salt corrections forreactions using different enzymes or concentrations of enzymes, or forany other variation made in reaction conditions, involves a two stepprocess of a) measuring reaction temperature optima under the newreaction conditions, and varying the salt concentration within the T_(m)algorithm to produce a calculated temperature matching or closelyapproximating the observed optima. Measurement of an optimum reactiontemperature generally involves performing reactions at a range oftemperatures selected such that the range allows observation of anincrease in performance as an optimal temperature is approached (eitherby increasing or decreasing temperatures), and a decrease in performancewhen an optimal temperature has been passed, thereby allowingidentification of the optimal temperature or temperature range (Seee.g., Lyamichev, et al., Biochemistry 39: 9523 [2000]).

[0372] In some embodiments, a secondary reaction is used where thereleased cleavage fragment from a primary reaction hybridizes to asynthetic cassette to form a secondary cleavage reaction. In somepreferred embodiments, the cassette comprises a fluorescing moiety and aquenching moiety, wherein cleavage of the secondary cleavage structureseparates the fluorescing moiety from the quenching moiety, resulting ina detectable signal (e.g., FRET detection). The secondary reaction canbe configured a number of different ways. For example, in someembodiments, the synthetic cassette comprises two oligonucleotides: anoligonucleotide that contains the FRET moieties and a FRET/INVADERoligonucleotide bridging oligonucleotide that allows the INVADERoligonucleotide (i.e., the released flap from the primary reaction) andthe FRET oligonucleotide to hybridize thereto, such that a cleavagestructure is formed. In some embodiments, the synthetic cassette isprovided as a single oligonucleotide, comprising a hairpin structure(i.e., the FRET oligonucleotide is connected at its 3′ end to thebridging oligonucleotide by a loop). The loop may be nucleic acid, or anon-nucleic acid spacer or linker. The linked molecules may together bedescribed as a FRET cassette. In the secondary reaction using a FRETcassette the released flap from the primary reaction, which acts as anINVADER oligonucleotide, should be able to associate and disassociatewith the FRET cassette freely, so that one released flap can direct thecleavage of multiple FRET cassettes. It is one aspect of the assaydesign that all of the probe sequences may be selected to allow theprimary and secondary reactions to occur at the same optimaltemperature, so that the reaction steps can run simultaneously. In analternative embodiment, the probes may be designed to operate atdifferent optimal temperatures, so that the reaction steps are notsimultaneously at their temperature optima. As noted above, the sameiterative process used to select the ASR of the probe can be used in thedesign of the portion of the primary probe that participates in asecondary reaction.

[0373] Another determinant of hybridization efficiency is the saltconcentration of the reaction. In large part, the choice of solutionconditions will depend on the requirements of the cleavage agent, andfor reagents obtained commercially, the manufacturer's instructions area resource for this information. When developing an assay utilizing anyparticular cleavage agent, the oligonucleotide and temperatureoptimizations described above should be performed in the bufferconditions best suited to that cleavage agent.

[0374] In some embodiments, additional agents may be included inreaction mixtures to enhance assay performance. For example, chargedcompounds such as aminoglycosides and other polyamines have been used tomodulate DNA and RNA conformation and function (see, e.g., Earnshaw andGait, Nucl. Acids Res. 26:5551 [1998]; Robinson and Wang, Nucl. AcidsRes. 24:676 [1996]; Jerinie, J. Mol. Biol. 304(5):707 [2000]; Schroederet al., EMBO 19(1):l [2000]). Inclusion of the aminoglycoside antibioticneomycin sulfate (e.g., at 1 μM in a primary reaction) can enhance assayperformance by, e.g., reducing background signal, and therefore reducingthe limit of detection of a particular INVADER assay probe set.Compounds of this type that may find use in INVADER assay reactionsinclude, but are not limited to, aminoglycosides, oligomerizedaminoglycosides, and aminoglycoside bioconjugates, and other polyanionsincluding, but not limited to, spermine and hexaamine cobalt.

[0375] A “no enzyme” control allows the assessment of the stability ofthe labeled oligonucleotides under particular reaction conditions, or inthe presence of the sample to be tested e.g., in assessing the samplefor contaminating nucleases). In this manner, the substrate andoligonucleotides are placed in a tube containing all reactioncomponents, except the enzyme and treated the same as theenzyme-containing reactions. Other controls may also be included. Forexample, a reaction with all of the components except the target nucleicacid will serve to confirm the dependence of the cleavage on thepresence of the target sequence.

[0376] d) Selection of a Cleavage Agent

[0377] As demonstrated in a number of the Examples, some 5′ nucleases donot require an upstream oligonucleotide to be active in a cleavagereaction. Although cleavage may be slower without the upstreamoligonucleotide, it may still occur (Lyamichev et al., Science 260:778[1993], Kaiser et al., J. Biol. Chem., 274:21387 [1999]). When a DNAstrand is the template or target strand to which probe oligonucleotidesare hybridized, the 5′ nucleases derived from DNA polymerases and someflap endonucleases (FENs), such as that from Methanococcus jannaschii,can cleave quite well without an upstream oligonucleotide providing anoverlap (Lyamichev et al., Science 260:778 [1993], Kaiser et al., J.Biol. Chem., 274:21387 [1999], and U.S. Pat. No. 5,843,669, hereinincorporated by reference in its entirety). These nucleases may beselected for use in some embodiments of the INVADER assay, e.g., inembodiments wherein cleavage of the probe in the absence of an INVADERoligonucleotide gives a different cleavage product, which does notinterfere with the intended analysis, or wherein both types of cleavage,INVADER oligonucleotide-directed and INVADERoligonucleotide-independent, are intended to occur.

[0378] In other embodiments it is preferred that cleavage of the probebe dependent on the presence of an upstream INVADER oligonucleotide, andenzyme having this requirement would be used. Other FENs, such as thosefrom Archeaoglobus fulgidus (Afu) and Pyrococcus furiosus (Pfu), cleavean overlapped structure on a DNA target at so much greater a rate thanthey do a non-overlapping structure (i.e., either missing the upstreamoligonucleotide or having a non-overlapping upstream oligonucleotide)that they can be viewed as having an essentially absolute requirementfor the overlap (Lyamichev et al., Nat. Biotechnol., 17:292 [1999],Kaiser et al., J. Biol. Chem., 274:21387 [1999]). When an RNA target ishybridized to DNA oligonucleotide probes to form a cleavage structure,many FENs cleave the downstream DNA probe poorly, regardless of thepresence of an overlap. On such an RNA-containing structure, the 5′nucleases derived from DNA polymerases have a strong requirement for theoverlap, and are essentially inactive in its absence. The selection ofenymes for use in the detection of RNA targets is discussed in moredetail below, in Section IV: Improved Enzymes For Use In INVADEROligonucleotide-Directed Cleavage Reactions Comprising RNA Targets.

[0379] e) Probing for Multiple Alleles

[0380] The INVADER oligonucleotide-directed cleavage reaction is alsouseful in the detection and quantification of individual variants oralleles in a mixed sample population. By way of example, such a needexists in the analysis of tumor material for mutations in genesassociated with cancers. Biopsy material from a tumor can have asignificant complement of normal cells, so it is desirable to detectmutations even when present in fewer than 5% of the copies of the targetnucleic acid in a sample. In this case, it is also desirable to measurewhat fraction of the population carries the mutation. Similar analysesmay also be done to examine allelic variation in other gene systems, andit is not intended that the method of the present invention by limitedto the analysis of tumors.

[0381] As demonstrated below, in one embodiment, reactions can beperformed under conditions that prevent the cleavage of probes bearingeven a single-nucleotide difference mismatch, but that permit cleavageof a similar probe that is completely complementary to the target inthis region. In a preferred embodiment, a mismatch is positioned at thenucleotide in the probe that is 5′ of the site where cleavage occurs inthe absence of the mismatch.

[0382] In other embodiments, the INVADER assay may be performed underconditions that have a tight requirement for an overlap (e.g., using theAfu FEN for DNA target detection or the 5′ nuclease of DNA polymerasefor RNA target detection, as described above), providing an alternativemeans of detecting single nucleotide or other sequence variations. Inone embodiment, the probe is selected such that the target basesuspected of varying is positioned at the 5′ end of thetarget-complementary region of this probe. The upstream INVADERoligonucleotide is positioned to provide a single base of overlap. Ifthe target and the probe oligonucleotide are complementary at the basein question, the overlap forms and cleavage can occur. However, if thetarget does not complement the probe at this position, that base in theprobe becomes part of a non-complementary 5′ arm, no overlap between theINVADER oligonucleotide and probe oligonucleotide exists, and cleavageis suppressed.

[0383] It is also contemplated that different sequences may be detectedin a single reaction. Probes specific for the different sequences may bedifferently labeled. For example, the probes may have different dyes orother detectable moieties, different lengths, or they may havedifferences in net charges of the products after cleavage. Whendifferently labeled in one of these ways, the contribution of eachspecific target sequence to final product can be tallied. This hasapplication in detecting the quantities of different versions of a genewithin a mixture. Different genes in a mixture to be detected andquantified may be wild type and mutant genes (e.g., as may be found in atumor sample, such as a biopsy). In this embodiment, one might designthe probes to precisely the same site, but one to match the wild-typesequence and one to match the mutant. Quantitative detection of theproducts of cleavage from a reaction performed for a set amount of timewill reveal the ratio of the two genes in the mixture. Such analysis mayalso be performed on unrelated genes in a mixture. This type of analysisis not intended to be limited to two genes. Many variants within amixture may be similarly measured.

[0384] Alternatively, different sites on a single gene may be monitoredand quantified to verify the measurement of that gene. In thisembodiment, the signal from each probe would be expected to be the same.

[0385] It is also contemplated that multiple probes may be used that arenot differently labeled, such that the aggregate signal is measured.This may be desirable when using many probes designed to detect a singlegene to boost the signal from that gene. This configuration may also beused for detecting unrelated sequences within a mix. For example, inblood banking it is desirable to know if any one of a host of infectiousagents is present in a sample of blood. Because the blood is discardedregardless of which agent is present, different signals on the probeswould not be required in such an application of the present invention,and may actually be undesirable for reasons of confidentiality.

[0386] Just as described for the two-oligonucleotide system, above, thespecificity of the detection reaction will be influenced by theaggregate length of the target nucleic acid sequences involved in thehybridization of the complete set of the detection oligonucleotides. Forexample, there may be applications in which it is desirable to detect asingle region within a complex genome. In such a case the set ofoligonucleotides may be chosen to require accurate recognition byhybridization of a longer segment of a target nucleic acid, often in therange of 20 to 40 nucleotides. In other instances it may be desirable tohave the set of oligonucleotides interact with multiple sites within atarget sample. In these cases one approach would be to use a set ofoligonucleotides that recognize a smaller, and thus statistically morecommon, segment of target nucleic acid sequence.

[0387] In one preferred embodiment, the INVADER and stackeroligonucleotides may be designed to be maximally stable, so that theywill remain bound to the target sequence for extended periods during thereaction. This may be accomplished through any one of a number ofmeasures well known to those skilled in the art, such as adding extrahybridizing sequences to the length of the oligonucleotide (up to about50 nts in total length), or by using residues with reduced negativecharge, such as phosphorothioates or peptide-nucleic acid residues, sothat the complementary strands do not repel each other to degree thatnatural strands do. Such modifications may also serve to make theseflanking oligonucleotides resistant to contaminating nucleases, thusfurther ensuring their continued presence on the target strand duringthe course of the reaction. In addition, the INVADER and stackeroligonucleotides may be covalently attached to the target (e.g., throughthe use of psoralen cross-linking).

[0388] f) Applications for Pooled DNA and RNA Samples

[0389] In some embodiments, the present invention provides methods andkits for assaying a pooled sample using INVADER detection reagents (e.g.primary probe, INVADER probe, and FRET cassette). In some preferredembodiments, the kit comprises instructions on how to perform theINVADER assay and specifically how to apply the INVADER detection assayto pooled samples from many individuals, or to “pooled” samples frommany cells (e.g. from a biopsy sample) from a single subject.

[0390] In particular embodiments, the present invention allows detectionof polymorphims in pooled samples combined from many individuals in apopulation (e.g. 10, 50, 100, or 500 individuals), or from a singlesubject where the nucleic acid sequences are from a large number ofcells that are assayed at once. In this regard, the present inventionallows the frequency of rare mutations in pooled samples to be detectedand an allele frequency for the population established. In someembodiments, this allele frequency may then be used to statisticallyanalyze the results of applying the INVADER detection assay to anindividual's frequency for the polymorphism (e.g. determined using theINVADER assay). In this regard, mutations that rely on a percent ofmutants found (e.g. loss of heterozygozity mutations) may be analyzed,and the severity of disease or progression of a disease determined (See,e.g. U.S. Pat. No. 6,146,828 and 6,203,993 to Lapidus, herebyincorporated by reference for all purposes, where genetic testing andstatistical analysis are employed to find disease causing mutations oridentify a patient sample as containing a disease causing mutations).

[0391] In some embodiments of the present invention, broad populationscreens are performed. In some preferred embodiments, pooling DNA fromseveral hundred or a thousand individuals is optimal. In such a pool,for example, DNA from any one individual would not be detectable, andany detectable signal would provide a measure of frequency of thedetected allele in a broader population. The amount of DNA to be used,for example, would be set not by the number of individuals in a pool,but rather by the allele frequency to be detected. For example, in someembodiments, an assay gives ample signal from 20 to 40 ng of DNA in a 90minute reaction. At this level of sensitivity, analysis of 1 μg of DNAfrom a high-complexity pool would produce comparable signal from allelespresent in only about 3-5% of the population.

[0392] g) Applications of RNA Detection.

[0393] RNA quantitation is becoming increasingly important in basic,pharmaceutical, and clinical research. For example, quantitation ofviral RNAs can predict disease progression and therapeutic efficacy.Likewise, gene expression analysis of diseased vs. normal, or untreatedvs. treated, tissue can identify relevant biological responses or assessthe effects of pharmacological agents. As the focus of the Human GenomeProject moves toward gene expression analysis, the field will require aflexible RNA analysis technology that can quantitatively monitormultiple forms of alternatively transcribed and/or processed RNAs.

[0394] As decribed above for the detection of multiple alleles,multiplex formats of the RNA INVADER assay enable simultaneousexpression analysis of two or more genes within the same sample. In aprimary reaction, one-nucleotide overlap-substrates are generated by thehybridization of INVADER oligonucleotides and probe oligonucleotides totheir respective RNA targets. Each probe contains a specific,target-complementary region and a distinctive non-complementary 5′ flapthat is associated only with that specific mRNA in that assay. Thedistinctive flaps may be distinguished in any of the myriad waysdisclosed herein (e.g., with different labels, different secondarycleavage systems having different labels, specific antibodies, differentsizes when resolved, differenct sequences detected by hybridization insolution or on surfaces, etc.)

[0395] While the RNA invasive cleavage assay, like the method used forDNA detection described above, can use two invasive cleavage reactionsin sequence (described below, in Section II of the Detailed Descriptionof the Invention), its preference for the 5′ nucleases derived from DNApolymerases (described in detail in Section IV of the DetailedDescription of the Invention) indicates that additional format changesare preferred. Unlike the FEN 5′ nucleases generally used for detectionof DNA targets, optimal signal amplification with the DNA Pol-related 5′nucleases occurs only when a probe turnover mechanism is employed inboth the primary and secondary reactions (in contrast to an INVADERoligonucleotide turnover mechanism, wherein an INVADER oligonucleotidecycles, e.g., to direct the cleavage of multiple FRET cassettes, asdescribed below, in Section II of this Detailed Description).Consequently, in preferred embodiments, RNA detection uses sequentialoperation of the two reactions, rather than simultaneous reactionperformance. Because the reactions are performed truly sequentially, inthese embodiments, the RNA INVADER assay signal accumulates linearly inboth a target- and time-dependent manner. In contrast, the primary andsecondary reactions of the DNA INVADER assay, when run concurrently,amplify signal as a linear function of target level, but as a quadraticfunction of time. In the sequential embodiments, the RNA INVADER assayuses two separate oligonucleotides, a secondary probe (e.g., a FRETprobe) and secondary target, for signal generation.

[0396] A key feature of the RNA invasive cleavage assay is its abilityto discriminate highly homologous RNA sequences, such as those found incytochrome P450 gene families. Like the DNA INVADER assay, the RNAINVADER assay can discriminate single-base changes. In some embodiments,the first 5′ complementary base of each probe is positioned at anon-conserved site in its mRNA target, so that a mismatch preventsformation of the overlap-structure, and thus prevents cleavage of theprobe. Alternatively spliced mRNA variants can be specifically detectedby positioning the cleavage site at a splice junction.

[0397] To monitor large changes in mRNA levels, the dynamic range of theassay can be extended using real-time analysis. However, since the assaygenerates signal linearly with time or target level, simply varying theamount of sample added per reaction and calculating the copies of mRNAper ng total RNA enables accurate quantitation with a single endpointmeasurement on low-cost instrumentation. Further, in cases whereabsolute quantitation is not necessary, the assay's linear signalamplification mechanism and reproducibility also eliminate the need fora standard curve and enable simple and precise relative quantitation ofany one gene.

[0398] The RNA INVADER assay is particularly suited for detectingalternatively spliced or edited RNA variants because even a single basechange at the overlap site affects 5′ nuclease cleavage. All areasrequiring RNA quantitation, such as high-throughput screening in drugdiscovery research, monitoring drug metabolism and safety in clinicaltrials, and clinical load monitoring of viral RNA can use thistechnology. Splice variants can be monitored in at least two ways withthe assay: 1) detection of an individual exon or 2) detection of aspecific splice junction.

[0399] To examine an RNA population for variants having more or fewerexons after splicing, INVADER assay probe sets are designed for each ofthe exons of interest (or for all exons in the mature RNA). Quantitationof exons, independent of how many mRNAs they reside in, may provideinformation about the number of splice variants for a given gene, aswell as indicate the levels of expression for each exon. Mini in vitrotranscripts containing only one or a few exons can be generated for eachprobe set so that absolute quantitation can be performed for each exon,thus enabling accurate comparisons of exon levels. If it is known that aparticular exon is present in all known variants, in some embodiments, aprobe set is designed for that exon for use as an internal control tonormalize across different samples. RNAs having a one copy of each exon(e.g., “normally spliced” RNA) should produce signal from the collectionof probe sets in certain relative amounts (which should be esseintiallyequal for all exons, corrected for variations in the sensitivity ofindividual probe sets; see Section V). Alterations in splicing alter therelative amounts of the exons. For example, if all of the produced RNAsare missing one of the normal exons, the signal for that exon dropstoward zero, while if half of the RNAs are missing that exon, the signalfor that exon drops toward 50%. More complex combinations of splicevariations and mixtures of differently spliced mRNAs yield more complexand more informative profiles. Detection is not limited to exons. RNApopulations may also be monitored for the presence of intron sequencesthat are usually removed by splicing. Such global exon screeningprovides biologically relevant or diagnostic information when comparingnormal vs. diseased tissue or untreated vs. treated cells (e.g., inpharmacogenomic screening assays). An array-based description of thistype of measurement is referred to as alternative splicing detectorarrays (D. Black, Cell 103:367 [2000]). It is contemplated that the mRNAINVADER assay gives similar results but with greater specificity andmore accurate quantitation than the oligonucleotide array formats.

[0400] In an alternative embodiment, alternatively spliced mRNAs isdetected by examiniation of specific splice junctions. The advantage ofmonitoring the splice sites, as opposed to the exons themselves, is thateven splice variants involving very small exons (e.g. <10 nts) areaccurately detected with the assay.

[0401] Additionally, in some embodiments, the mRNA INVADER assay isalsoused to monitor alternative start and stop sites in the mRNA, and isused to monitor lifetimes of processed and unprocessed RNAs and RNAfragments (e.g., as used in timecourse studies following induction).

[0402] II. Signal Enhancement by Incorporating the Products of anInvasive Cleavage Reaction into a Subsequent Invasive Cleavage Reaction

[0403] As noted above, the oligonucleotide product released by theinvasive cleavage can be used subsequently in any reaction or read-outmethod that uses oligonucleotides in the size range of a cleavageproduct. In addition to the reactions involving primer extension andtranscription, described herein, another enzymatic reaction that makesuse of oligonucleotides is the invasive cleavage reaction. The presentinvention provide means of using the oligonucleotide released in aprimary invasive cleavage reaction as a component to complete a cleavagestructure to enable a secondary invasive cleavage reaction. IT is notintended that the sequential use of the invasive cleavage product belimited to a single additional step. It is contemplated that manydistinct invasive cleavage reactions may be performed in sequence.

[0404] The polymerase chain reaction uses a DNA replication method tocreate copies of a targeted segment of nucleic acid at a logarithmicrate of accumulation. This is made possible by the fact that when thestrands of DNA are separated, each individual strand contains sufficientinformation to allow assembly of a new complementary strand. When thenew strands are synthesized the number of identical molecules hasdoubled. Within 20 iterations of this process, the original may becopied 1 million-fold, making very rare sequences easily detectable. Themathematical power of a doubling reaction has been incorporated into anumber of amplification assays.

[0405] By performing multiple, sequential invasive cleavage reactionsthe method of the present invention captures an exponential mathematicaladvantage without producing additional copies of the target analyte. Ina simple invasive cleavage reaction the yield, Y, is simply the turnoverrate, K, multiplied by the time of the reaction, t (i.e., Y=(K)(t)). IfY is used to represent the yield of a simple reaction, then the yield ofa compound (i.e., a multiple, sequential reaction), assuming that eachof the individual invasive cleavage steps has the same turnover rate,can be simply represented as Y^(n), where n is the number of invasivecleavage reactions that have been performed in the series. If the yieldsof each step differ the ultimate yield can be represented as the productof the multiplication of the yields of each individual reaction in theseries. For example, if a primary invasive cleavage reaction can produceone thousand products in 30 minutes, and each of those products can inturn participate in 1000 additional reactions, there will be 1000²copies (1000×1000) of the ultimate product in a second reaction. If athird reaction is added to the series, then the theoretical yield willbe 1000³ (1000×1000×1000). In the methods of the present invention theexponent comes from the number of invasive cleavage reactions in thecascade. This can be contrasted to the amplification methods describedabove (e.g., PCR) in which Y is limited to 2 by the number of strands induplex DNA, and the exponent n is the number of cycles performed, sothat many iterations are necessary to accumulate large amounts ofproduct.

[0406] To distinguish the exponential amplifications described abovefrom those of the present invention, the former can be consideredreciprocating reactions because the products the reaction feed back intothe same reaction (e.g., event one leads to some number of events 2, andeach event 2 leads back to some number of events 1). In contrast, theevents in some embodiments of the present invention are sequential(e.g., event 1 leads to some number of events 2; each event 2 leads tosome number of events 3, etc., and no event can contribute to an eventearlier in the chain).

[0407] The sensitivity of the reciprocating methods is also one of thegreatest weaknesses when these assays are used to determine if a targetnucleic acid sequence is present or absent in a sample. Because theproduct of these reactions is detectable copies of the startingmaterial, contamination of a new reaction with the products of anearlier reaction can lead to false positive results, (i.e., the apparentdetection of the target nucleic acid in samples that do not actuallycontain any of that target analyte). Furthermore, because theconcentration of the product in each positive reaction is so high,amounts of DNA sufficient to create a strong false positive signal canbe communicated to new reactions very easily either by contact withcontaminated instruments or by aerosol. In contrast to the reciprocatingmethods, the most concentrated product of the sequential reaction (i.e.,the product released in the ultimate invasive cleavage event) is notcapable of initiating a like reaction or cascade if carried over to afresh test sample. This is a marked advantage over the exponentialamplification methods described above because the reactions of thepresent invention may be performed without the costly containmentarrangements (e.g., either by specialized instruments or by separatelaboratory space) required by any reciprocating reaction. While theproducts of a penultimate event may be inadvertently transferred toproduce a background of the ultimate product in the absence of the atarget analyte, the contamination would need to be of much greatervolume to give an equivalent risk of a false positive result.

[0408] When the term sequential is used it is not intended to limit theinvention to configurations in which that one invasive cleavage reactionor assay must be completed before the initiation of a subsequentreaction for invasive cleavage of a different probe. Rather, the termrefers to the order of events as would occur if only single copies ofeach of the oligonucleotide species were used in an assay. The primaryinvasive cleavage reaction refers to that which occurs first, inresponse to the formation of the cleavage structure on the targetnucleic acid. Subsequent reactions may be referred to as secondary,tertiary and so forth, and may involve artificial “target” strands thatserve only to support assembly of a cleavage structure, and which areunrelated to the nucleic acid analyte of interest. While the completeassay may, if desired, be configured with each step of invasive cleavageseparated either in space (e.g., in different reaction vessels) or intime (e.g., using a shift in reaction conditions, such as temperature,enzyme identity or solution condition, to enable the later cleavageevents), it is also contemplated that all of the reaction components maybe mixed so that secondary reactions may be initiated as soon as productfrom a primary cleavage becomes available. In such a format, primary,secondary and subsequent cleavage events involving different copies ofthe cleavage structures may take place simultaneously.

[0409] Several levels of this sort of linear amplification can beenvisioned, in which each successive round of cleavage produces anoligonucleotide that can participate in the cleavage of a differentprobe in subsequent rounds. The primary reaction would be specific forthe analyte of interest with secondary (and tertiary, etc.) reactionsbeing used to generate signal while still being dependent on the primaryreaction for initiation.

[0410] The released product may perform in several capacities in thesubsequent reactions. For example, the product of one invasive cleavagereaction becomes the INVADER oligonucleotide to direct the specificcleavage of another probe in a second reaction. In such an example, thefirst invasive cleavage structure is formed by the annealing of theINVADER oligonucleotide and the probe oligonucleotide (Probe 1) to thefirst target nucleic acid (Target 1). The target nucleic acid is dividedinto three regions based upon which portions of the INVADER and probeoligonucleotides are capable of hybridizing to the target. Region 1 ofthe target has complementarity to only the INVADER oligonucleotide;region 3 of the target has complementarity to only the probe; and region2 of the target has an overlap between the INVADER and probeoligonucleotides.

[0411] Cleavage of Probe 1 releases the “Cut Probe 1”. The releasedProbe 1 is then used as the INVADER oligonucleotide in second cleavage.The second cleavage structure is formed by the annealing of the CutProbe 1, a second probe oligonucleotide (“Probe 2”) and a second targetnucleic acid (“Target 2”). In some embodiments, Probe 2 and the secondtarget nucleic acid are covalently connected, preferably at their 3′ and5′ ends, respectively, thus forming a hairpin stem and loop, termedherein a “cassette”. The loop may be nucleic acidor a non-nucleic acidspacer or linker. Inclusion of an excess of the cassette molecule allowseach Cut Probe 1 to serve as an INVADER to direct the cleavage ofmultiple copies of the cassette.

[0412] Probe 2 may be labeled and detection of cleavage of the secondcleavage structure may be accomplished by detecting the labeled cutProbe 2; the label may a radioisotope (e.g., ³²P, ³⁵S), a fluorophore(e.g., fluorescein), a reactive group capable of detection by asecondary agent (e.g., biotin/streptavidin), a positively charged adductwhich permits detection by selective charge reversal (as discussed inSection IV above), etc. Alternatively, the cut Probe 2 may used in atailing reaction, or to complete or activate a protein-binding site, ormay be detected or used by any of the means for detecting or using anoligonucleotide described herein.

[0413] In other embodiments, probe oligonucleotides that are cleaved inthe primary reaction can be designed to fold back on themselves (i.e.,they contain a region of self-complementarity) to create a molecule thatcan serve as both the INVADER and target oligonucleotide (termed here an“IT” complex). The IT complex then enables cleavage of a different probepresent in the secondary reaction. Inclusion of an excess of thesecondary probe molecule (“Probe 2”), allows each IT molecule to serveas the platform for the generation of multiple copies of cleavedsecondary probe. The target nucleic acid is divided into three regionsbased upon which portions of the INVADER and probe oligonucleotides arecapable of hybridizing to the target (as discussed above and it is notedthat the target may be divided into four regions if a stackeroligonucleotide is employed). The second cleavage structure is formed bythe annealing of the second probe (“Probe 2”) to the fragment of Probe 1(“Cut Probe 1”) that was released by cleavage of the first cleavagestructure. The Cut Probe 1 forms a hairpin or stem/loop structure nearits 3′ terminus by virtue of the annealing of the regions ofself-complementarity contained within Cut Probe 1 (this self-annealedCut Probe 1 forms the IT complex). The IT complex (Cut Probe 1) isdivided into three regions. Region 1 of the IT complex hascomplementarity to the 3′ portion of Probe 2; region 2 hascomplementarity to both the 3′ end of Cut Probe 1 and to the 5′ portionof Probe 2; and region 3 contains the region of self-complementarity(i.e., region 3 is complementary to the 3′ portion of the Cut Probe 1).Note that with regard to the IT complex (i.e., Cut Probe 1), region 1 islocated upstream of region 2 and region 2 is located upstream of region3. As for other embodiments of invasive cleavage, region “2” canrepresent a region where there is a physical, but not sequence, overlapbetween the INVADER oligonucleotide portion of the Cut Probe 1 and theProbe 2 oligonucleotide.

[0414] The cleavage products of the secondary invasive cleavage reaction(i.e., Cut Probe 2) can either be detected, or can in turn be designedto constitute yet another integrated INVADER-target complex to be usedwith a third probe molecule, again unrelated to the preceding targets.

[0415] It is envisioned that the oligonucleotide product of a primarycleavage reaction may fill the role of any of the oligonucleotidesdescribed herein (e.g., it may serve as a target strand without anattached INVADER oligonucleotide-like sequence, or it may serve as astacker oligonucleotide, as described above), to enhance the turnoverrate seen in the secondary reaction by stabilizing the probehybridization through coaxial stacking.

[0416] Secondary cleavage reactions in some preferred embodiments of thepresent invention include the use of FRET cassettes. Such moleculesprovide both a secondary target and a FRET labeled cleavable sequence,allowing homogeneous detection (i.e., without product separation orother manipulation after the reaction) of the sequential invasivecleavage reaction. Other preferred embodiments use a secondary reactionsystem in which the FRET probe and synthetic target are provided asseparate oligonucleotides.

[0417] In a preferred embodiment, each subsequent reaction isfacilitated by (i.e., is dependent upon) the product of the previouscleavage, so that the presence of the ultimate product may serve as anindicator of the presence of the target analyte. However, cleavage inthe second reaction need not be dependent upon the presence of theproduct of the primary cleavage reaction; the product of the primarycleavage reaction may merely measurably enhance the rate of the secondcleavage reaction.

[0418] In summary, the INVADER assay cascade (i.e., sequential invasivecleavage reactions) of the present invention is a combination of two ormore linear assays that allows the accumulation of the ultimate productat an exponential rate, but without significant risk of carryovercontamination. It is important to note that background that does notarise from sequential cleavage, such as thermal breakage of thesecondary probe, generally increases linearly with time. In contrast,signal generation from a 2-step sequential reaction follows quadratickinetics. Thus, collection of data as a time course, either by takingtime points or through the use of an instrument that allows real-timedetection during the INVADER assay reaction incubations, provides theattractive capability of discriminating between the true signal and anybackground solely on the basis of quadratic versus linear increases insignal over time. For example, when viewed graphically, the real signalwill appear as a quadratic curve, while any accumulating background willbe linear, and thus easy to distinguish, even if the absolute level ofthe background signal (e.g., fluorescence in a FRET detection format) issubstantial.

[0419] The sequential invasive cleavage amplification of the presentinvention can be used as an intermediate boost to any of the detectionmethods (e.g., gel based analysis by either standard or by chargereversal), polymerase tailing, and incorporation into a protein bindingregion, described herein. When used is such combinations the increasedproduction of a specific cleavage product in the invasive cleavage assayreduces the burdens of sensitivity and specificity on the read-outsystems, thus facilitating their use.

[0420] In addition to enabling a variety of detection platforms, thecascade strategy is suitable for multiplex analysis of individualanalytes (i.e., individual target nucleic acids) in a single reaction.The multiplex format can be categorized into two types. In one case, itis desirable to know the identity (and amount) of each of the analytesthat can be present in a clinical sample, or the identity of each of theanalytes as well as an internal control. To identify the presence ofmultiple individual analytes in a single sample, several distinctsecondary amplification systems may be included. Each probe cleaved inresponse to the presence of a particular target sequence (or internalcontrol) can be designed to trigger a different cascade coupled todifferent detectable moieties, such as different sequences to beextended by DNA polymerase or different dyes in an FRET format. Thecontribution of each specific target sequence to final product canthereby be tallied, allowing quantitative detection of different genesor alleles in a sample containing a mixture of genes or alleles.

[0421] In the second configuration, it is desirable to determine if anyof several analytes are present in a sample, but the exact identity ofeach does not need to be known. For example, in blood banking it isdesirable to know if any one of a host of infectious agents is presentin a sample of blood. Because the blood is discarded regardless of whichagent is present, different signals on the probes would not be requiredin such an application of the present invention, and may actually beundesirable for reasons of confidentiality. In this case, the 5′ arms(i.e., the 5′ portion that will be released upon cleavage) of thedifferent analyte-specific probes would be identical and would thereforetrigger the same secondary signal cascade. A similar configuration wouldpermit multiple probes complementary to a single gene to be used toboost the signal from that gene or to ensure inclusivity when there arenumerous alleles of a gene to be detected.

[0422] In the primary INVADER assay reaction, there are two potentialsources of background. The first is from INVADERoligonucleotide-independent cleavage of probe annealed to the target, toitself, or to one of the other oligonucleotides present in the reaction.The use of an enzyme that cannot efficiently cleave a structure thatlacks a primer is preferred for this reason. The enzyme Pfu FEN-1 givesno detectable cleavage in the absence of the upstream oligonucleotide oreven in the presence of an upstream oligonucleotide that fails to invadethe probe-target complex. This indicates that the Pfu FEN-1 endonucleaseis a suitable enzyme for use in the methods of the present invention.

[0423] Other structure-specific nucleases may be suitable as a well. Asdiscussed in the first example, some 5′ nucleases can be used inconditions that significantly reduce this primer-independent cleavage.For example, it has been shown that when the 5′ nuclease of DNAPTaq isused to cleave hairpins the primer-independent cleavage is markedlyreduced by the inclusion of a monovalent salt in the reaction(Lyamichev, et al., [1993], supra).

[0424] III. Effect of ARRESTOR Molecules on Signal and Background inSequential Invasive Cleavage Reactions.

[0425] As described above, the concentration of the probe that iscleaved can be used to increase the rate of signal accumulation, withhigher concentrations of probe yielding higher final signal. However,the presence of large amounts of residual uncleaved probe can presentproblems for subsequent use of the cleaved products for detection or forfurther amplification. If the subsequent step is a simple detection(e.g., by gel resolution), the excess uncut material may causebackground by streaking or scattering of signal, or by overwhelming adetector (e.g., over-exposing a film in the case of radioactivity, orexceeding the quantitative detection limits of a fluorescence imager).This can be overcome by partitioning the product from the uncut probe.In more complex detection methods, the cleaved product may be intendedto interact with another entity to indicate cleavage. As noted above,the cleaved product can be used in any reaction that makes use ofoligonucleotides, such as hybridization, primer extension, ligation, orthe direction of invasive cleavage. In each of these cases, the fate ofthe residual uncut probe should be considered in the design of thereaction. In a primer extension reaction, the uncut probe can hybridizeto a template for extension. If cleavage is required to reveal thecorrect 3′ end for extension, the hybridized uncut probe will not beextended. It may, however, compete with the cleaved product for thetemplate. If the template is in excess of the combination of cleaved anduncleaved probe, then both of the latter should be able to find a copyof template for binding. If, however, the template is limiting, anycompetition may reduce the portion of the cleaved probe that can findsuccessfully bind to the available template. If a vast excess of probewas used to drive the initial reaction, the remainder may also be invast excess over the cleavage product, and thus may provide a veryeffective competitor, thereby reducing the amount of the final reaction(e.g., extension) product for ultimate detection.

[0426] The participation of the uncut probe material in a secondaryreaction can also contribute to background in these reactions. While thepresentation of a cleaved probe for a subsequent reaction may representan ideal substrate for the enzyme to be used in the next step, someenzymes may also be able to act, albeit inefficiently, on the uncutprobe as well. It was shown during the development of the presentinvention that transcription can be promoted from a nicked promoter evenwhen one side of the nick has additional unpaired nucleotides.Similarly, when the subsequent reaction is to be an invasive cleavage,the uncleaved probe may bind to the elements intended to form the secondcleavage structure with the cleaved probe. In experiments conductedduring the development of the present invention, it was found that someof the 5′ nucleases described herein can catalyze some measure ofcleavage of defective structures. Even at a low level, this aberrantcleavage can be misinterpreted as positive target-specific cleavagesignal.

[0427] With these negative effects of the surfeit of uncut probeconsidered, there is clearly a need for some method of preventing theseinteractions. As noted above, it is possible to partition the cleavedproduct from the uncut probe after the primary reaction by traditionalmethods. However, these methods are often time consuming, may beexpensive (e.g., disposable columns, gels, etc.), and may increase therisk for sample mishandling or contamination. It is far preferable toconfigure the sequential reactions such that the original sample neednot be removed to a new vessel for subsequent reaction.

[0428] The present invention provides a method for reducing interactionsbetween the primary probe and other reactants. This method provides ameans of specifically diverting the uncleaved probes from participationin the subsequent reactions. The diversion is accomplished by theinclusion in the next reaction step an agent designed to specificallyinteract with the uncleaved primary probe. While the primary probe in aninvasive cleavage reaction is discussed for reasons of convenience, itis contemplated that the ARRESTOR molecules may be used at any reactionstep within a chain of invasive cleavage steps, as needed or desired forthe design of an assay. It is not intended that the ARRESTOR moleculesof the present invention be limited to any particular step.

[0429] The method of diverting the residual uncut probes from a primaryreaction makes use of agents that can be specifically designed orselected to bind to the uncleaved probe molecules with greater affinitythan to the cleaved probes, thereby allowing the cleaved probe speciesto effectively compete for the elements of the subsequent reaction, evenwhen the uncut probe is present in vast excess. These agents have beentermed “ARRESTOR molecules,” due to their function of stopping orarresting the primary probe from participation in the later reaction. Invarious Examples below, an oligonucleotide is provided as an ARRESTORmolecule in an invasive cleavage assay. It can be appreciated that anymolecule or chemical that can discriminate between the full-length uncutprobe and the cleaved probe, and that can bind or otherwise disable theuncleaved probe preferentially may be configured to act as ARRESTORmolecules within the meaning of the present invention. For example,antibodies can be derived with such specificity, as can the “aptamers”that can be selected through multiple steps of in vitro amplification(e.g., “SELEX,” U.S. Pat. Nos. 5,270,163 and 5,567,588; hereinincorporated by reference) and specific rounds of capture or otherselection means.

[0430] In one embodiment, the ARRESTOR molecule is an oligonucleotide.In another embodiment the ARRESTOR oligonucleotides is a compositeoligonucleotide, comprising two or more short oligonucleotides that arenot covalently linked, but that bind cooperatively and are stabilized byco-axial stacking. In a preferred embodiment, the oligonucleotide ismodified to reduce interactions with the cleavage agents of the presentinvention. When an oligonucleotide is used as an ARRESTORoligonucleotide, it is intended that it not participate in thesubsequent reactive step. The binding of the ARRESTOR oligonucleotide tothe primary probe may, either with the participation of the secondarytarget, or without such participation, create a bifurcated structurethat is a substrate for cleavage by the 5′ nucleases used in someembodiments of the methods of the present invention. Formation of suchstructures would lead to some level of unintended cleavage that couldcontribute to background, reduce specific signal or compete for theenzyme. It is preferable to provide ARRESTOR oligonucleotides that willnot create such cleavage structures. One method of doing this is to addto the ARRESTOR oligonucleotides such modifications as have been foundto reduce the activity of INVADER oligonucleotides, as the INVADERoligonucleotides occupy a similar position within a cleavage structure(i.e., the 3′ end of the INVADER oligonucleotide positions the site ofcleavage of an unpaired 5′ arm). Modification of the 3′ end of theINVADER oligonucleotides was examined for the effects on cleavage in theExample section below; a number of the modifications tested were foundto be significantly debilitating to the function of the INVADERoligonucleotide. Other modifications not described herein may be easilycharacterized by performing such a test using the cleavage enzyme to beused in the reaction for which the ARRESTOR oligonucleotide is intended.

[0431] In a preferred embodiment, the backbone of an ARRESTORoligonucleotide is modified. This may be done to increase the resistanceto degradation by nucleases or temperature, or to provide duplexstructure that is a less favorable substrate for the enzyme to be used(e.g., A-form duplex vs. B-form duplex). In particularly preferredembodiment, the backbone-modified oligonucleotide further comprises a 3′terminal modification. In a preferred embodiment, the modificationscomprise 2′ O-methyl substitution of the nucleic acid backbone, while ina particularly preferred embodiment, the 2′ O-methyl modifiedoligonucleotide further comprises a 3′ terminal amine group.

[0432] The purpose of the ARRESTOR oligonucleotide is to allow theminority population of cleaved probe to effectively compete with theuncleaved probe for binding whatever elements are to be used in the nextstep. While an ARRESTOR oligonucleotide that can discriminate betweenthe two probe species absolutely (i.e., binding only to uncut and neverto cut) may be of the greatest benefit in some embodiments, it isenvisioned that in many applications, including the sequential INVADERassays described herein, the ARRESTOR oligonucleotide of the presentinvention may perform the intended function with only partialdiscrimination. When the ARRESTOR oligonucleotide has some interactionwith the cleaved probe, it may prevent detection of some portion ofthese cleavage products, thereby reducing the absolute level of signalgenerated from a given amount of target material. If this same ARRESTORoligonucleotide has the simultaneous effect of reducing the backgroundof the reaction (i.e., from non-target specific cleavage) by a factorthat is greater than the factor of reduction in the specific signal,then the significance of the signal (i.e., the ratio of signal tobackground), is increased, even with the lower amount of absolutesignal. Any potential ARRESTOR molecule design may be tested in a simplefashion by comparing the levels of background and specific signals fromreactions that lack ARRESTOR molecules to the levels of background andspecific signal from similar reactions that include ARRESTORoligonucleotides. What constitutes an acceptable level of tradeoff ofabsolute signal for specificity will vary for different applications(e.g., target levels, read-out sensitivity, etc.), and can be determinedby any individual user using the methods of the present invention. Insome embodiments, an ARRESTOR oligonucleotide may be used to stop signalgeneration from one or more particular target nucleic acids (e.g., aninternal standard, or one that is present in higher abundance) so thatincubation of the reaction mixture can be continued such that additionalsignal can accumulate from one or more other target nucleic acids in thesame reaction vessel.

[0433] IV. Improved Enzymes for use in INVADER Oligonucleotide-DirectedCleavage Reactions Comprising RNA Targets;

[0434] A cleavage structure is defined herein as a structure that isformed by the interaction of a probe oligonucleotide and a targetnucleic acid to form a duplex, the resulting structure being cleavableby a cleavage agent, including but not limited to an enzyme. Thecleavage structure is further defined as a substrate for specificcleavage by the cleavage means in contrast to a nucleic acid moleculethat is a substrate for nonspecific cleavage by agents such asphosphodiesterases. In considering improvements to enzymatic cleavageagents, one may consider the action of said enzymes on any structuresthat fall within the definition of a cleavage structure. Specificcleavage at any site within such a structure is contemplated.

[0435] Improvements in an enzyme may be an increased or decreased rateof cleavage of one or more types of structures. Improvements may alsoresult in more or fewer sites of cleavage on one or more of saidcleavage structures. In developing a library of new structure-specificnucleases for use in nucleic acid cleavage assays, improvements may havemany different embodiments, each related to the specific substratestructure used in a particular assay.

[0436] As an example, one embodiment of the INVADERoligonucleotide-directed cleavage assay of the present invention may beconsidered. In the INVADER oligonucleotide-directed cleavage assay, theaccumulation of cleaved material is influenced by several features ofthe enzyme behavior. Not surprisingly, the turnover rate, or the numberof structures that can be cleaved by a single enzyme molecule in a setamount of time, is very important in determining the amount of materialprocessed during the course of an assay reaction. If an enzyme takes along time to recognize a substrate (e.g., if it is presented with aless-than-optimal structure), or if it takes a long time to executecleavage, the rate of product accumulation is lower than if these stepsproceeded quickly. If these steps are quick, yet the enzyme “holds on”to the cleaved structure, and does not immediately proceed to anotheruncut structure, the rate will be negatively affected.

[0437] Enzyme turnover is not the only way in which enzyme behavior cannegatively affect the rate of accumulation of product. When the meansused to visualize or measure product is specific for a precisely definedproduct, products that deviate from that definition may escapedetection, and thus the rate of product accumulation may appear to belower than it is. For example, if one had a sensitive detector fortrinucleotides that could not see di- or tetranucleotides, or any sizedoligonucleotide other that 3 residues, in the INVADER-directed cleavageassay of the present invention any errant cleavage would reduce thedetectable signal proportionally. It can be seen from the cleavage datapresented here that, while there is usually one site within a probe thatis favored for cleavage, there are often products that arise fromcleavage one or more nucleotides away from the primary cleavage site.These are products that are target-dependent, and are thus notnon-specific background. Nevertheless, if a subsequent visualizationsystem can detect only the primary product, these represent a loss ofsignal. One example of such a selective visualization system is thecharge reversal readout presented herein, in which the balance ofpositive and negative charges determines the behavior of the products.In such a system the presence of an extra nucleotide or the absence ofan expected nucleotide can exclude a legitimate cleavage product fromultimate detection by leaving that product with the wrong balance ofcharge. It can be easily seen that any assay that can sensitivelydistinguish the nucleotide content of an oligonucleotide, such asstandard stringent hybridization, suffers in sensitivity when somefraction of the legitimate product is not eligible for successfuldetection by that assay.

[0438] These discussions suggest two highly desirable traits in anyenzyme to be used in the method of the present invention. First, themore rapidly the enzyme executes an entire cleavage reaction, includingrecognition, cleavage and release, the more signal it may potentiallycreated in the INVADER oligonucleotide-directed cleavage assay. Second,the more successful an enzyme is at focusing on a single cleavage sitewithin a structure, the more of the cleavage product can be successfullydetected in a selective read-out.

[0439] The rationale cited above for making improvements in enzymes tobe used in the INVADER oligonucleotide-directed cleavage assay are meantto serve as an example of one direction in which improvements might besought, but not as a limit on either the nature or the applications ofimproved enzyme activities. As another direction of activity change thatwould be appropriately considered improvement, the DNAP-associated 5′nucleases may be used as an example. In creating some of thepolymerase-deficient 5′ nucleases described herein it was found that thethose that were created by deletion of substantial portions of thepolymerase domain, assumed activities that were weak or absent in theparent proteins. These activities included the ability to cleavenon-forked structures, a greatly enhanced ability to exonucleolyticallyremove nucleotides from the 5′ ends of duplexed strands, and a nascentability to cleave circular molecules without benefit of a free 5′ end.

[0440] In addition to the 5′ nucleases derived from DNA polymerases, thepresent invention also contemplates the use of structure-specificnucleases that are not derived from DNA polymerases. For example, aclass of eukaryotic and archaebacterial endonucleases have beenidentified which have a similar substrate specificity to 5′ nucleases ofPol I-type DNA polymerases. These are the FEN1 (Flap EndoNuclease),RAD2, and XPG (Xeroderma Pigmentosa-complementation group G) proteins.These proteins are involved in DNA repair, and have been shown to favorthe cleavage of structures that resemble a 5′ arm that has beendisplaced by an extending primer during polymerization. Similar DNArepair enzymes have been isolated from single cell and higher eukaryotesand from archaea, and there are related DNA repair proteins ineubacteria. Similar 5′ nucleases have also been associated withbacteriophage such as T5 and T7.

[0441] Recently, the 3-dimensional structures of DNAPTaq and T5 phage5′-exonuclease were determined by X-ray diffraction (Kim et al., Nature376:612 [1995]; and Ceska et al., Nature 382:90 [1995]). The two enzymeshave very similar 3-dimensional structures despite limited amino acidsequence similarity. The most striking feature of the T5 5′-exonucleasestructure is the existence of a triangular hole formed by the activesite of the protein and two alpha helices. This same region of DNAPTaqis disordered in the crystal structure, indicating that this region isflexible, and thus is not shown in the published 3-dimensionalstructure. However, the 5′ nuclease domain of DNAPTaq is likely to havethe same structure, based its overall 3-dimensional similarity to T55′-exonuclease, and that the amino acids in the disordered region of theDNAPTaq protein are those associated with alpha helix formation. Theexistence of such a hole or groove in the 5′ nuclease domain of DNAPTaqwas predicted based on its substrate specificity (Lyamichev et al.,supra).

[0442] It has been suggested that the 5′ arm of a cleavage structuremust thread through the helical arch described above to position saidstructure correctly for cleavage (Ceska et al., supra). One of themodifications of 5′ nucleases described herein opened up the helicalarch portion of the protein to allow improved cleavage of structuresthat cut poorly or not at all (e.g., structures on circular DNA targetsthat would preclude such threading of a 5′ arm). The gene construct thatwas chosen as a model to test this approach was the one called CLEAVASEBN, which was derived from DNAPTaq but does not contain the polymerasedomain. It comprises the entire 5′ nuclease domain of DNAP Taq, and thusshould be very close in structure to the T5 5′ exonuclease. This 5′nuclease was chosen to demonstrate the principle of such a physicalmodification on proteins of this type. The arch-opening modification ofthe present invention is not intended to be limited to the 5′ nucleasedomains of DNA polymerases, and is contemplated for use on anystructure-specific nuclease that includes such an aperture as alimitation on cleavage activity. The present invention contemplates theinsertion of a thrombin cleavage site into the helical arch of DNAPsderived from the genus Thermus as well as 5′ nucleases derived fromDNAPs derived from the genus Thermus. The specific example shown hereinusing the CLEAVASE BN/thrombin nuclease merely illustrates the conceptof opening the helical arch located within a nuclease domain. As theamino acid sequence of DNAPs derived from the genus Thermus are highlyconserved, the teachings of the present invention enable the insertionof a thrombin site into the helical arch present in these DNAPs and 5′nucleases derived from these DNAPs.

[0443] The opening of the helical arch was accomplished by insertion ofa protease site in the arch. This allowed post-translational digestionof the expressed protein with the appropriate protease to open the archat its apex. Proteases of this type recognize short stretches ofspecific amino acid sequence. Such proteases include thrombin and factorXa. Cleavage of a protein with such a protease depends on both thepresence of that site in the amino acid sequence of the protein and theaccessibility of that site on the folded intact protein. Even with acrystal structure it can be difficult to predict the susceptibility ofany particular region of a protein to protease cleavage. Absent acrystal structure it must be determined empirically.

[0444] In selecting a protease for a site-specific cleavage of a proteinthat has been modified to contain a protease cleavage site, a first stepis to test the unmodified protein for cleavage at alternative sites. Forexample, DNAPTaq and CLEAVASE BN nuclease were both incubated underprotease cleavage conditions with factor Xa and thrombin proteases. Bothnuclease proteins were cut with factor Xa within the 5′ nuclease domain,but neither nuclease was digested with large amounts of thrombin. Thus,thrombin was chosen for initial tests on opening the arch of theCLEAVASE BN enzyme.

[0445] In the protease/CLEAVASE modifications described herein thefactor Xa protease cleaved strongly in an unacceptable position in theunmodified nuclease protein, in a region likely to compromise theactivity of the end product. Other unmodified nucleases contemplatedherein may not be sensitive to the factor Xa, but may be sensitive tothrombin or other such proteases. Alternatively, they may be sensitiveto these or other such proteases at sites that are immaterial to thefunction of the nuclease sought to be modified. In approaching anyprotein for modification by addition of a protease cleavage site, theunmodified protein should be tested with the proteases underconsideration to determine which proteases give acceptable levels ofcleavage in other regions.

[0446] Working with the cloned segment of DNAPTaq from which theCLEAVASE BN protein is expressed, nucleotides encoding a thrombincleavage site were introduced in-frame near the sequence encoding aminoacid 90 of the nuclease gene. This position was determined to be at ornear the apex of the helical arch by reference to both the 3-dimensionalstructure of DNAPTaq, and the structure of T5 5′ exonuclease. Theencoded amino acid sequence, LVPRGS, was inserted into the apex of thehelical arch by site-directed mutagenesis of the nuclease gene. Theproline (P) in the thrombin cleavage site was positioned to replace aproline normally in this position in CLEAVASE BN because proline is analpha helix-breaking amino acid, and may be important for the3-dimensional structure of this arch. This construct was expressed,purified and then digested with thrombin. The digested enzyme was testedfor its ability to cleave a target nucleic acid, bacteriophage M13genomic DNA, that does not provide free 5′ ends to facilitate cleavageby the threading model.

[0447] While the helical arch in this nuclease was opened by proteasecleavage, it is contemplated that a number of other techniques could beused to achieve the same end. For example, the nucleotide sequence couldbe rearranged such that, upon expression, the resulting protein would beconfigured so that the top of the helical arch (amino acid 90) would beat the amino terminus of the protein, the natural carboxyl and aminotermini of the protein sequence would be joined, and the new carboxylterminus would lie at natural amino acid 89. This approach has thebenefit that no foreign sequences are introduced and the enzyme is asingle amino acid chain, and thus may be more stable that the cleaved 5′nuclease. In the crystal structure of DNAPTaq, the amino and carboxyltermini of the 5′-exonuclease domain lie in close proximity to eachother, which suggests that the ends may be directly joined without theuse of a flexible linker peptide sequence as is sometimes necessary.Such a rearrangement of the gene, with subsequent cloning and expressioncould be accomplished by standard PCR recombination and cloningtechniques known to those skilled in the art.

[0448] The INVADER invasive cleavage reaction has been shown to beuseful in the detection of RNA target strands (See e.g., U.S. Pat. No.6,001,567, incorporated herein by reference in its entirety). As withthe INVADER assay for the detection of DNA (Lyamichev et al., Nat.Biotechnol., 17:292 [1999]), the reactions may be run under conditionsthat permit the cleavage of many copies of a probe for each copy of thetarget RNA present in the reaction. In one embodiment, the reaction maybe performed at a temperature close to the melting temperature (T_(m))of the probe that is cleaved, such that the cleaved and uncleaved probesreadily cycle on and off the target strand without temperature cycling.Each time a full-length probe binds to the target in the presence of theINVADER oligonucleotide, it may be cleaved by a 5′ nuclease enzyme,resulting in an accumulation of cleavage product. The accumulation ishighly specific for the sequence being detected, and may be configuredto be proportional to both time and target concentration of thereaction. In another embodiment, the temperature of the reaction may beshifted (i.e., it may be raised to a temperature that will cause theprobe to dissociate) then lowered to a temperature at which a new copyof the probe hybridizes to the target and is cleaved by the enzyme. In afurther embodiment, the process of raising and lowering the temperatureis repeated many times, or cycled, as it is in PCR (Mullis and Faloona,Methods in Enzymology, 155:335 [1987], Saiki et al., Science 230:1350[1985]).

[0449] As noted above, 5′ nucleases of Pol A type DNA polymerases arepreferred for cleavage of an invasive cleavage structure that comprisesan RNA target strand. The present invention provides enzymes havingimproved performance in detection assays based on the cleavage of astructure comprising RNA. In particular, the altered polymerases of thepresent invention exhibit improved performance in detection assays basedon the cleavage of a DNA member of an invasive cleavage structure thatcomprises an RNA target strand.

[0450] The 5′ nucleases of the present invention may be derived from PolA type DNA polymerases. The terminology used in describing thealterations made in this class of 5′ nucleases relates to thedescriptions of DNA polymerase structures known in the art. The Klenowfragment of the Pol A polymerase from E. coli (the C-terminal twothirds, which has the DNA synthesizing activity but lacks the 5′nuclease activity) has been described as having a physical formresembling a right hand, having an open region called the “palm”, and acleft that holds the primer/template duplex defined on one side by a“fingers” domain and on the other by a “thumb” domain (Joyce and Steitz,Trends in Biochemical Science 12:288 [1987]). This is shownschematically in FIG. 5. Because this physical form has proved to becommon to all Pol A DNA polymerases and to a number of additionaltemplate-dependent polymerizing enzymes such as reverse transcriptases,the hand terminology has become known in the art, and the sites ofactivity in these enzymes are often described by reference to theirposition on the hand. For reference, and not intended as a limitation onthe present invention, the palm is created from roughly the first 200amino acids of the polymerase domain, the thumb from the middle 140, andthe fingers by the next 160, with the base of the cleft formed from theremaining regions (FIGS. 6). Although some enzymes may deviate fromthese structural descriptions, the equivalent domains and sequencescorresponding to such domains may be identified by sequence homology toknown enzyme sequences, by comparison of enzyme crystal structures, andother like methods.

[0451] In creating the improved enzymes of the present invention,several approaches have been taken, although the present invention innot limited to any particular approach. First two DNA polymerases, Taqand Tth, that have different rates of DNA strand cleavage activity onRNA targets were compared. To identify domains related to thedifferences in activity, a series of chimerical constructs was createdand the activities were measured. This process identified two regions ofthe Tth polymerase that could, if transferred into the Taq polymerase,confer on the TaqPol an RNA-dependent cleavage activity equivalent tothat of the native Tth protein. Once these regions were identified, theparticular amino acids involved in the activity were examined. Since thetwo proteins are about 87 percent identical in amino acid sequenceoverall, the identified regions had only a small number of amino aciddifferences. By altering these amino acids singly and in combinations, apair of amino acids were identified in TthPol that, if introduced intothe TaqPol protein, increased the rate of cleavage up to that of thenative TthPol.

[0452] These data demonstrate two important aspects of the presentinvention. First, specific amino acids can be changed to conferTthPol-like RNA-dependent cleavage activity on a polymerase having alesser activity. More broadly, however, these results provide regions ofthese polymerases that are involved in the recognition of theRNA-containing cleavage structure. Identification of these importantregions, combined with published information on the relationships ofother amino acids to the various functions of these DNA polymerases andcomputer-assisted molecular modeling during the development of thepresent invention have allowed a rational design approach to createadditional improved 5′ nucleases. The information also allowed a focusedrandom mutagenesis approach coupled with a rapid screening procedure toquickly create and identify enzymes having improved properties. Usingthese methods of the present invention, a wide array of improvedpolymerases are provided.

[0453] The methods used in creating and selecting the improved 5′nucleases of the present invention are described in detail below and inthe experimental examples. A general procedure for screening andcharacterizing the cleavage activity of any 5′ nuclease is included inthe experimental examples. The methods discussions are divided into thefollowing sections: I) Creation and selection of chimerical constructs;II) Site-specific mutagenesis based on information from chimericalconstructs; III) Site-specific mutagenesis based on molecular modelingand published physical studies; and IV) focused random mutagenesis.Additional chimerical constructs not included in this discussion aredescribed in the Experimental Examples.

[0454] 1) Creation and Selection of Chimerical Constructs

[0455] The PolA-type DNA polymerases, including but not limited to DNApolymerase enzymes from Thermus species, comprise two distinctivedomains, the 5′ nuclease and the polymerase domains, shown schematicallyin FIG. 6. The polymerase domains reside in the C-terminal two-thirds ofthe proteins and are responsible for both DNA-dependent andRNA-dependent DNA polymerase activities. The N-terminal one-thirdportions contain the 5′ nuclease domains. In the genus Thermus Pol Apolymerase, the palm region consists of, roughly, amino acids 300-500,the thumb region includes amino acids 500-650, while the fingers regionis formed by the remaining amino acids from 650 to 830 (FIG. 6).

[0456] The derivatives, Taq DN RX HT and Tth DN RX HT, of Taq and TthPolused in many of the experiments of the present invention, and describedherein, are modified to reduce synthetic activity and to facilitatechimera construction, but have 5′ nuclease activity essentiallyidentical to unmodified TaqPol and TthPol. Unless otherwise specified,the TaqPol and TthPol enzymes of the following discussion refer to theDN RX HT derivative.

[0457] TthPol has a 4-fold higher cleavage rate with the IL-6 RNAtemplate (shown in FIG. 10) than TaqPol (shown in FIGS. 11 and 12),although the Taq and TthPols show similarities of cleavage in DNA targetstructures (FIG. 10). Since the amino acid sequences of TaqPol andTthPol (FIGS. 8 and 9) share about 87% identity and greater than 92%similarity, the high degree of homology between the enzymes allowedcreation of a series of chimeric enzymes between TthPol and TaqPol. Theactivity of the chimeric enzymes was used as a parameter to identify theregion(s) of these proteins affecting RNA dependent 5′ nucleaseactivity.

[0458] The chimeric constructs between TthPol and TaqPol genes shownschematically in FIGS. 7 and 19 were created by swapping DNA fragmentsdefined by the restriction endonuclease sites, EcoRI and BamHI, commonfor both genes, the cloning vector site SalI and the new sites, NotI,BstBI and NdeI, created at the homologous positions of both genes bysite directed mutagenesis. The restriction enzymes have been abbreviatedas follows: EcoRI is E; NotI is N; BstBI is Bs; NdeI is D, BamHI is B,and SalI is S.

[0459] The activity of each chimeric enzyme was evaluated using theinvasive signal amplification assay with the IL-6 RNA target (FIG. 10),and the cycling cleavage rates shown in FIG. 12 were determined asdescribed in the Experimental Examples. Comparison of the cleavage ratesof the first two chimeras, TaqTth(N) and TthTaq(N), created by swappingthe polymerase and 5′ nuclease domains at the NotI site (FIG. 7), showsthat TaqTth(N) has the same activity as TthPol, whereas its counterpartTthTaq(N) retains the activity of TaqPol (FIG. 12). This resultindicates that the higher cleavage rate of TthPol is associated with itspolymerase domain and suggests an important role of the polymerasedomain in the 5′ nuclease activity.

[0460] The next step was to identify a minimal region of TthPolpolymerase that would give rise to the TthPol-like RNA dependent 5′nuclease activity when substituted for the corresponding region of theTaqPol sequence. To this end, the TaqTth(N) chimera was selected togenerate a series of new constructs by replacing its TthPol sequencewith homologous regions of TaqPol. First, the N-terminal and C-terminalparts of the TaqPol polymerase domain were substituted for thecorresponding regions of TaqTth(N) using the common BamHI site as abreaking point to create TaqTth(N-B) and TaqTth(B-S) chimeras,respectively (FIG. 7). TaqTth(N-B) which has the TthPol sequence betweenamino acids 328 and 593, is approximately 3 times more active than theTaqTth(B-S) and 40% more active than TthPol (FIG. 12). This resultestablishes that the NotI-BamHI portion of the TthPol polymerase domaindetermines superior RNA-dependent 5′ nuclease activity of TthPol.

[0461] From these data it was determined that a central portion of theTthPol, when used to replace the homologous portion of TaqPol(TaqTth(N-B) construct) could confer superior RNA recognition on thechimerical protein composed primarily of Taq protein. In fact, thecycling rate of this chimerical protein exceeded that of the parentTthPol. Comparison of chimeras that included sub-portions of theactivity-improving region of TthPol, approximately 50% of the region ineach case (See, TaqTth(N-D) and TaqTth(D-B), FIGS. 7 and 12) showed nosignificant improvement in RNA dependent activity as compared to theparent TaqPol. This result indicates that aspects of each half of theregion are required for this activity. A construct having an onlyslightly smaller portion of the Tth insert portion (TaqTth(Bs-B)) showedactivity that was close to that of the parent TthPol protein, but whichwas less than that of the TaqTth(N-B) construct.

[0462] 2) Site-Specific Mutagenesis Based on Information from ChimericalConstructs

[0463] Comparison of the TthPol and TaqPol amino acid sequences betweenthe BstBI and BamHI sites reveals only 25 differences (FIG. 13). Amongthose, there are 12 conservative changes and 13 substitutions resultingin a change in charge. Since the analysis of the chimeric enzymes hassuggested that some critical amino acid changes are located in bothBstBI-NdeI and NdeI-BamHI regions of TthPol, site directed mutagenesiswas used to introduce the TthPol specific amino acids into theBstBI-NdeI and NdeI-BamHI regions of the TaqTth(D-B) and TaqTth (N-D)chimeras, respectively. Six TthPol-specific substitutions were generatedin the BstBI-NdeI region of the TaqTth(D-B) by single or double aminoacid mutagenesis and only one double mutation, W417L/G418K, was able torestore the TthPol activity with the IL-6 RNA target (See e.g., FIG.14). Similarly, 12 TthPol specific amino acids were introduced at thehomologous positions of the NdeI-BamHI region of the TaqTth(N-D) andonly one of them, E507Q, increased the cleavage rate to the TthPol level(See e.g., FIG. 14).

[0464] To confirm that the W417L, G418K and E507Q substitutions aresufficient to increase the TaqPol activity to the TthPol level, TaqPolvariants carrying these mutations were created and their cleavage rateswith the IL-6 RNA substrate were compared with that of TthPol. FIG. 15shows that the TaqPol W417L/G418K/E507Q and TaqPol G418K/E507Q mutantshave 1.4 times higher activity than TthPol and more than 4 fold higheractivity than TaqPol, whereas the TaqPol W417L/E507Q mutant has the sameactivity as TthPol, which is about 3 fold higher than TaqPol. Theseresults demonstrate that K418 and Q507 of TthPol are important aminoacids in defining its superior RNA dependent 5′ nuclease activitycompared to TaqPol.

[0465] The ability of these amino acids to improve the RNA dependent 5′nuclease activity of a DNA polymerase was tested by introducing thecorresponding mutations into the polymerase A genes of two additionalorganisms: Thermus filiformus and Thermus scotoductus. TaqPol showedimproved RNA dependent activity when it was modified to contain theW417L and E507Q mutations, which made it more similar at these residuesto the corresponding residues of TthPol (K418 and Q507). The TfiPol wasmodified to have P420K and E507Q, creating TfiDN 2M, while the TscPolwas modified to have E416K and E505Q, to create TscDN 2M. The activityof these enzymes for cleaving various DNA and RNA containing structureswas determined as described in Example 1, using the IdT2, IrT3, hairpinand X-structures diagrammed in FIGS. 21 and 22, with the results shownin both FIG. 25 and Table 8. Both enzymes have much less RNA-dependentcleavage activity than either the TthPol or the Taq 2M enzymes. However,introduction of the mutations cited above into these polymerasesincreased the RNA dependent cleavage activity over 2 fold compared tothe unmodified enzymes (FIG. 25). These results demonstrate thattransferability of improved RNA dependent cleavage activity into a widerange of polymerases using the methods of the present invention.

[0466] 3) Site-Specific Mutagenesis Based on Molecular Modeling andPublished Physical Studies

[0467] The positions of the G418H and E507Q mutations in the crystalstructure of a complex of the large fragment of TaqPol (Klentaq1) with aprimer/template DNA determined by Li et al. (Li et al., Protein Sci.,7:1116 [1998]) are shown in FIG. 17. The E507Q mutation is located atthe tip of the thumb subdomain at a nearest distance of 3.8 A and 18 Afrom the backbone phosphates of the primer and template strands,respectively. The interaction between the thumb and the minor groove ofthe DNA primer/template was previously suggested by the co-crystalstructures of Klenow fragment DNA polymerase I (Breese et al., Science260:352 [1993]) and TaqPol (Eom et al, Nature 382:278 [1996]). Deletionof a 24 amino acid portion of the tip of the thumb in Klenow fragment,corresponding to amino acids 494-518 of TaqPol, reduces the DNA bindingaffinity by more than 100-fold (Minnick et al., J. Biol. Chem.,271:24954 [1996]). These observations are consistent with the hypothesisthat the thumb region, which includes the E507 residue, is involved ininteractions with the upstream substrate duplex.

[0468] The W417L and the G418K mutations in the palm region of TaqPol(FIG. 17) are located approximately 25 A from the nearest phosphates ofthe template and upstream strands, according to the co-crystalstructures of TaqPol with duplex DNA bound in the polymerizing mode (Liet al., Protein Sci., 7:1116 [1998], Eom et al., Nature 382:278 [1996]).The same distance was observed between the analogous W513 and P514 aminoacids of Klenow fragment and the template strand of DNA bound in theediting mode (Breese et al., Science 260:352 [1993]). Thus, nointeractions between TaqPol and the overlapping substrate can besuggested from the available co-crystal studies for this region.

[0469] Although an understanding of the mechanism of action of theenzymes is not necessary for the practice of the present invention andthe present invention is not limited to any mechanism of action, it isproposed that the amino acids at positions 417 and 418 in the palmregion of TaqPol interact with the upstream substrate duplex only whenthe enzyme functions as a 5′ nuclease, but no interaction with theseamino acids occurs when TaqPol switches into polymerizing mode. Thishypothesis suggests a novel mode of substrate binding by DNA polymerasescalled here the “5′ nuclease mode.” Several lines of evidence supportthis hypothesis. The study of the chimeric enzymes described hereclearly separates regions of the polymerase domain involved in the 5′nuclease and polymerase activities. Accordingly, the W417L and G418Kmutations, together with the E507Q mutation, affect the 5′ nucleaseactivity of TaqPol on substrates having an RNA target strand (FIG. 15),but have no effect on either RNA-dependent or DNA-dependent DNApolymerase activities (FIG. 16). On the other hand, mutations in theactive site of TaqPol, such as R573A, R587A, E615A, R746A, N750A andD785N, which correspond to substitutions in Klenow fragment of E.coliDNA Pol I that affect both polymerase activity and substrate bindingaffinity in the polymerizing mode (Polesky et al., J. Biol. Chem.,265:14579 [1990], Polesky et al., J. Biol. Chem., 267:8417 [1992],Pandey et al., Eur. J. Biochem., 214:59 [1993]) were shown to havelittle or no effect on the 5′ nuclease activity. Superposition of thepolymerase domains of TaqPol (Eom et al., Nature 382:278 [1996]), E.coliPol I and Bacillus stearothermophilus Pol I (Kiefer et al., Nature391:304 [1998]) using the programs DALI (Holm and Sander, J. Mol. Biol.,233:123 [1993], Holm and Sander, Science 273:595 [1996]) and Insight II(Molecular Simulation Inc., Naperville, Ill.) shows that the palm regionof TaqPol between amino acids 402-451, including W417 and G418, isstructurally highly conserved between the three polymerases, althoughthere is no structural similarity between the rest of the palmsubdomains. This observation suggests an important role for this regionin eubacterial DNA polymerases.

[0470] The 5′ nuclease and polymerase activities should be preciselysynchronized to create a nicked structure rather than a gap or anoverhang that could cause a deletion or an insertion during Okazakifragment processing or DNA repair, if ligase joins the endsinappropriately. According to the previously proposed model (Kaiser etal., J. Biol. Chem., 274:21387 [1999]), the 3′ terminal nucleotide ofthe upstream strand is sequestered by the 5′ nuclease domain to preventits extension, thus halting synthesis. The interaction with the 3′nucleotide apparently activates the 5′ nuclease that endonucleoliticallyremoves the displaced 5′ arm of the downstream strand. This cleavageoccurs by the precise incision at the site defined by the 3′ nucleotide,thus creating the nick. This model requires a substantial rearrangementof the substrate-enzyme complex, which may include a translocation ofthe complex to the 5′ nuclease mode to separate the primer/template fromthe polymerase active site.

[0471] It is possible that a relocation of the substrate away from thepolymerase active site could be induced by the interaction between theduplex formed between the template and incoming strands and the creviceformed by the finger and thumb subdomains. Such an interaction couldforce conformational transitions in the thumb that would bring thetemplate/primer duplex into close contact with the W417 and G418 aminoacids. Significant flexibility of the thumb has been previously reportedthat might explain such changes (Beese et al., Science 260:352 [1993],Eom et al., Nature 382:278 [1996], Ollis et al., Nature 313:762 [1985],Kim et al., Nature 376:612 [1995], Korolev et al., Proc. Natl. Acad.Sci., 92:9264 [1995], Li et al, EMBO J., 17:7514 [1998]). Additionalconformational changes of the fingers domain that might help to open thecrevice, such as the transition from the ‘closed’ to the ‘open’structure described by Li et al. (Li et al., EMBO J., 17:7514 [1998]),are consistent with this model. It may be that the 5′ nuclease bindingmode was not observed in any of the published co-crystal structures of aDNA Pol I because the majority of the structures were solved for thepolymerase domain only, with a template/primer substrate rather thanwith an overlapping 5′ nuclease substrate.

[0472] K_(m) values of 200-300 nM have been determined for TaqPol,TthPol and TaqPol G418K/E507Q for the RNA containing substrate. Thesevalues are much higher than the K_(m) value of <1 nM estimated forTthPol with an all-DNA overlapping substrate suggesting that the RNAtemplate adversely affects substrate binding. The low affinity could beexplained by the unfavorable interaction between the enzyme and eitherthe A-form duplex adopted by the substrate with an RNA target, or theribose 2′ hydroxyls of the RNA strand. Between these two factors, thelatter seems more likely, since the 5′ nucleases of eubacterial DNApolymerases can efficiently cleave substrates with an RNA downstreamprobe (Lyamichev et al., Science 260:778 [1993]), which would presumablyhave an A-form. Further, the co-crystal studies suggest that thetemplate/primer duplex partially adopts a conformation close to A-formin its complex with DNA polymerase (Eom et al., Nature 382:278 [1996],Kiefer et al., Nature 391:304 [1998], Li et al., EMBO J., 17:7514[1998]). The G418K/E507Q mutations increase the k_(cat) of TaqPol morethan two fold, but have little effect on K_(m). Such an effect would beexpected if the mutations position the substrate in an orientation moreappropriate for cleavage rather than simply increasing the bindingconstant.

[0473] In addition to the mutational analysis described above, anotherapproach to studying specific regions of enzymes, enzymestructure-function relationships, and enzyme-substrate interaction is toinvestigate the actual, physical structure of the molecule.

[0474] With the advances in crystallographic, NMR, and computer andsoftware technology, study of molecular structure has become a viabletool for those interested in the configuration, organization, anddynamics of biomolecules. Molecular modeling has increased theunderstanding of the nature of the interactions that underlie thestructure of proteins and how proteins interact functionally withsubstrate. Numerous publications describing the structures of variouspolymerases or polymerase protein portions, HIV reverse transcriptase,and other nucleic acid binding proteins have provided mechanisticinsights into protein conformation, changes in conformation, andmolecular interactions necessary for function.

[0475] As an example, the report by Doublie et al. (Doublie et al.,Nature 391:251 [1998]) discloses the crystal structure of T7 DNApolymerase and provides information about which amino acid regions arelikely to have an affect on substrate binding, which are required tocontact the substrate for polymerization, and which amino acids bindcofactors, such as metal ions. It is noted in this paper and others thatmany of the polymerases share not only sequence similarity, butstructural homology as well. When certain structural domains ofdifferent polymerases are superimposed (for example, T7 polymerase,Klenow fragment editing complex, the unliganded Taq DNA polymerase andthe Taq Polymerase-DNA complex) conserved motifs are clearlydiscernable.

[0476] Specifically, combining the information from all of thesedifferent structural sources and references, a model of the proteininteracting with DNA, RNA, or heteroduplex can be made. The model canthen be examined to identify amino acids that may be involved insubstrate recognition or substrate contact. Changes in amino acids canbe made based on these observations, and the effects on the variousactivities of the 5′ nuclease proteins are assessed using screeningmethods such as those of the present invention, described in theexperimental examples.

[0477] The domain swapping analysis discussed previously demonstratedthat sequences of TthDN that are important in RNA-dependent 5′ nucleaseactivity lie in the polymerase domain of the protein. Therefore, studyof structural data of the polymerase domain with respect to nucleic acidrecognition provides one method of locating amino acids that, whenaltered, alter RNA recognition in a 5′ nuclease reaction. For example,analysis conducted during the development of the present inventionexamined published analyses relating to primer/template binding by thepolymerase domain of E. coli Pol 1, the Klenow fragment. Table 2 shows asampling of kinetic constants determined for the Klenow fragment, andshows the effects a number of mutations on these measurements. Thecorresponding or similarly positioned amino acids in the TaqPol areindicated in the right hand column. It was postulated that mutationshaving a noticible impact on the interactions of the Klenow fragmentwith the DNA template or the primer/template duplex, as indicated bychanges in K_(d) and Relative DNA affinity values, might also haveeffects when made at the corresponding sites in TaqPol and relatedchimerical or mutant derivatives. A selection of the mutations thatproduced a higher K_(d) value or a lower Relative DNA affinity valuewhen introduced into the Klenow fragment were created and examined inTaqPol. These Taq derivatives include, but are not limited to, thoseindicated by asterisks in the right hand column of Table 2.

[0478] For some Klenow variants, such as the R682 mutants, selection fortesting was not made based on the DNA affinity measurements, but becausemolecular modeling suggested interaction between some aspect of thetemplate/primer duplex and that amino acid. Similarly, additionalregions of Taq polymerase (or Taq derivatives) were targeted formutagenesis based on structural data and information from molecularmodeling. Based on modeling, the thumb region was postulated to contactan RNA template. Thus, amino acids in the thumb region were looked forthat, if altered, might alter that contact. For example, FIGS. 6 and 17show that amino acids 502, 504, and 507 are located at the tip of thethumb. It was postulated that altering these amino acids might have anaffect on the enzyme-substrate interaction. Using the activity screeningmethods described In Example 1, mutations that produced beneficialeffects were identified. This approach was used to create a number ofimproved enzymes. For example, TaqPol position H784, corresponding toKlenow amino acid H881, is an amino acid in the fingers region and, assuch, may be involved in primer/template substrate binding. When theH881 amino acid in the Klenow enzyme is replaced by alanine, the changedecreases the affinity of the enzyme for DNA to only 30 to 40% of thewild type level. An analogous substitution was tested in aTaqPol-derived enzyme. Starting with the Taq derivativeW417L/G418K/E507Q, amino acid 784 was changed from Histidine (H) toAlanine (A) to yield the W417L/G418K/E507Q/H784A mutant, termed Taq 4M.This variant showed improved 5′ nuclease activity on the RNA test IrT(FIG. 24) test substrate (data in Table 3). Amino acid R587 is in thethumb region, and was selected for mutation based on its close proximityto the primer/template duplex in computer models. When an R587A mutationwas added to the Taq 4M variant, the activity on the test IrT1 testsubstrate was still further improved. In addition, the reduction,relative to the 4M variant, in cleavage of the X structure shown in FIG.22 constitutes an additional improvement in this enzyme's function.

[0479] Not all amino acid changes that reduce DNA binding in thepolymerization affect the 5′ nuclease activity. For example, mutationsE615A, R677A, affecting amino acid that are also in the thumb andfingers domains, respectively, have either adverse effect, or no effecton the 5′ nuclease activities, respectively, as measured using the testsubstrates in FIGS. 21 and 22, and compared to the parent variants thatlacked these changes. The R677A mutation was added to, and compared withthe TaqSS variant, while the E615A mutation was added to and comparedwith the Taq 4M variant. The test methods described herein provide aconvenient means of analyzing any variant for the alterations in thecleavage activity of both invasive an noninvasive substrates, for bothDNA and RNA containing structures. Thus, the present invention providesmethods for identifying all suitable improved enzymes.

[0480] Alterations that might increase the affinity of the enzymes forthe nucleic acid targets were also examined. Many of the mutationsdescribed above were selected because they caused the Klenow fragmentenzyme to have decreased affinity for DNA, with the goal of creatingenzymes more accepting of structures containing non-DNA strands. Ingeneral, the native DNA polymerases show a lower affinity for RNA/DNAduplexes, compared to their affinity for DNA/DNA duplexes. During thedevelopment of the present invention, it was sought to increase thegeneral affinity of the proteins of the present invention for a nucleicacid substrate without restoring or increasing any preference forstructures having DNA rather than RNA target strands. The substitutionof amino acids having different charges was examined as a means ofaltering the interaction between the proteins and the nucleic acidsubstrates. For example, it was postulated that addition of positivelycharged amino acid residues, such as lysine (K), might increase theaffinity of a protein for a negatively charged nucleic acid.

[0481] As noted above, alterations in the thumb region could affect theinteractions of the protein with the nucleic acid substrate. In oneexample, the mutation G504K (tip of the thumb) was introduced in Taq4Mand caused and enhancement of nuclease activity by 15% on an RNA target.Additional positively charged mutations (A502K and E507K) furtherimprove the RNA target dependent activity by 50% compared to the parentTaq4M enzyme.

[0482] The use of data from published studies and molecular modeling, incombination with results accrued during the development of the presentinvention allowed the identification of regions of the proteins in whichchanges of amino acids would be likely to cause observable differencesin at least one aspect of cleavage function. While regions could betargeted in this way, it was observed that changes in different aminoacids, even if near or immediate neighbors in the protein, could havedifferent effects. For example, while the A502K substitution created amarked increase in the RNA-dependent cleavage activity of Taq 4M,changing amino acid 499 from G to a K, only 3 amino acids away from 502,gave minimal improvement. As can be seen in the Experimental Examples,the approach of the present invention was to change several amino acidsin a candidate region, either alone or in combination, then use thescreening method provided in Example 1 to rapidly assess the effects ofthe changes. In this way, the rational design approach is easily appliedto the task of protein engineering.

[0483] In addition to the thumb, palm, and hand regions found in thepolymerase domain of these proteins, regions that are specific to 5′nucleases and nuclease domains were examined. Comparative studies on avariety of 5′ nucleases have shown that, though the amino acid sequencesvary dramatically from enzyme to enzyme, there are structural featurescommon to most. Two of these features are the helix-hairpin-helix motif(H-h-H) and the arch or loop structure. The H-h-H motif is believed tomediate non-sequence specific DNA binding. It has been found in at least14 families of proteins, including nucleases, N-glycosylases, ligases,helicases, topoisomerases, and polymerases (Doherty et al., Nucl. Acid.Res., 24:2488 [1996]). The crystallographic structure of rat DNApolymerase pol β bound to a DNA template-primer shows non-specifichydrogen bonds between the backbone nitrogens of the pol β HhH motif andthe phosphate oxygens of the primer of the DNA duplex (Pelletier et al,Science 264:1891 [1994]). Because the HhH domain of 5′ nuclease domainsof Taq and Tth polymerases may function in a similar manner, it iscontemplated that mutations in the HhH region of the enzyme alteractivity. Mutations may be introduced to alter the shape and structureof the motif, or to change the charge of the motif to cause increased ordecreased affinity for substrate.

[0484] Another structure common to many 5′ nucleases from diversesources such as eukaryotes, eubacteria, archaea and phage, is the archor loop domain. The crystal structure of the 5′ exonuclease ofbacteriophage T5 showed a distinct arch formed by two helices, onepositively charged and one containing hydrophobic residues (Ceska etal., Nature 382:90 [1996]). Interestingly, three residues that areconserved between T5 and Taq, Lys 83, Arg 86 and Tyr 82 are all in thearch. These correspond to amino acids Lys 83, Arg 86, and Tyr 82 in TaqDNA polymerase. The crystal structure for Taq (5′ nuclease) has alsobeen determined (Kim et al., Nature 376:612 [1995]).

[0485] The crystal structure from the flap endonuclease-1 fromMethanococcus janneschii also shows such a loop motif (Hwang et al.,Nat. Struct. Biol., 5:707 [1998]). The backbone crystal structure of MjaFEN-1 molecules may be superimposed on T5 exonuclease, Taq5′-exonuclease and T4 RNase H. An interesting feature common to all ofthese is the long loop. The loop of FEN-1 consists of a number ofpositively charged and aromatic residues and forms a hole withdimensions large enough to accommodate a single-stranded DNA molecule.The corresponding region in T5 exonuclease consists of three helicesforming a helical arch. The size of the hole formed by the helical archin T5 exonuclease is less than half that formed by the LI loop in MjFEN-1. In T4 RNase H or Taq 5′ exonuclease, this region is disordered.Some regions of the arch bind metals, while other regions of the archcontact nucleic acid substrate. Alignment of the amino-acid sequences ofsix 5′ nuclease domains from DNA polymerases in the pol I family showsix highly conserved sequence motifs containing ten conserved acidicresidues (Kim et al., Nature 376 [1995]).

[0486] The effects of alterations in the arch region were examined. InTaq polymerase the arch region is formed by amino acids 80-95 and96-109. Site directed mutagenesis was performed on the arch region.Alignment of amino acid sequences of the FEN and polymerase 5′ nucleasessuggested the design of 3 amino acid substitution mutations, P88E, P90Eand G80E. These substitutions were made on the Taq4M polymerase mutantas a parent enzyme. Results indicated that although the backgroundactivity on the HP and X substrates shown in FIG. 22 are tremendouslysuppressed in all mutants, the desirable 5′ nuclease activity on propersubstrates (IdT and IrT, FIG. 24) is also reduced. Despite the sequencehomology between Taq and Tth polymerases, they have very differentactivity on HP and X substrates. The alignment of the Taq and Tthpolymerase arch regions also demonstrates regions of extensive sequencehomology as well as minor differences. These differences led to thedesign of mutations L109F and A110T using Taq4M to generate Taq4ML109F/A110T, and the mutant Taq 4M A502K/G504K/E507K/T514S to generateTaq 4M L109F/A110T/A502K/G504K/E507K/T514S mutant. These two mutationshave drastically converted Taq4M enzyme to become more like Tth enzymein terms of the background substrate specificity while the 5′ nucleaseactivities on both DNA and RNA substrates are almost unchanged.

[0487] 4) Focused Random Mutagenesis

[0488] As described above, physical studies and molecular modeling maybe used alone or in combination to identify regions of the enzymes inwhich changes of amino acids are likely to cause observable differencesin at least one aspect of cleavage function. In the section above, useof this information was described to select and change specific aminoacids or combinations of amino acids. Another method of generating anenzyme with altered function is to introduce mutations randomly. Suchmutations can be introduced by a number of methods known in the art,including but not limited to, PCR amplification under conditions thatfavor nucleotide misincorporation (REF), amplification using primershaving regions of degeneracy (i.e., base positions in which differentindividual, but otherwise similar oligonucleotides in a reaction mayhave different bases), and chemical synthesis. Many methods of randommutagenesis are known in the art (Del R10 et al., Biotechniques 17:1132[1994]), and may be incorporated into the production of the enzymes ofthe present invention. The discussions of any particular means ofmutagenesis contained herein are presented solely by way of example andnot intended as a limitation. When random mutagenesis is performed suchthat only a particular region of an entire protein is varied, it can bedescribed as “focused random mutagenesis.” As described in theExperimental Examples, a focused random mutagenesis approach was appliedto vary the HhH and the thumb domains some of the enzyme variantspreviously created. These domains were chosen to provide examples ofthis approach, and it is not intended that the random mutagenesisapproach be limited to any particular domain, or to a single domain. Itmay be applied to any domain, or to any entire protein. Proteins thusmodified were tested for cleavage activity in the screening reactionsdescribed in Example 1, using the test substrates diagrammed in FIGS. 22and 24, with the results described in Tables 6 and 7.

[0489] Random mutagenesis was performed on the HhH region with theparent TaqSS or TthDN H785A mutants. None of the 8 mutants generatedshowed an improvement in activity compared to the parent enzyme (Table6). In fact, mutations of the region between residues 198-205 have about2-5 fold lower activity on both DNA and RNA substrates, suggesting thatthis region is essential for substrate recognition. Mutagenesis in thethumb region resulted in new mutations that improved 5′ nucleaseactivity by 20-100% on a DNA target and about 10% on an RNA target(Table 7).

[0490] Numerous amino acids in each of the distinct subdomains playroles in substrate contact. Mutagenesis of these may alter substratespecificity by altering substrate binding. Moreover, mutationsintroduced in amino acids that do not directly contact the substrate mayalso alter substrate specificity through longer range or generalconformation altering effects. These mutations may be introduced by anyof several methods known in the art, including, but not limited torandom mutagenesis, site directed mutagenesis, and generation ofchimeric proteins.

[0491] As noted above, numerous methods of random mutagenesis are knownin the art. The methods applied in the focused random mutagenesisdescribed herein may be applied to whole genes. It is also contemplatedthat additional useful chimerical constructs may be created through theuse of molecular breeding (See e.g., U.S. Pat. No. 5,837,458 and PCTPublications WO 00/18906, WO 99/65927, WO 98/31837, and WO 98/27230,herein incorporated by reference in their entireties). Regardless of themutagenesis method chosen, the rapid screening method described hereinprovides a fast and effective means of identifying beneficial changeswithin a large collection of recombinant molecules. This makes therandom mutagenesis procedure a manageable and practical tool forcreating a large collection of altered 5′ nucleases having beneficialimprovements. The cloning and mutagenesis strategies employed for theenzymes used as examples are applicable to other thermostable andnon-thermostable Type A polymerases, since DNA sequence similarity amongthese enzymes is very high. Those skilled in the art would understandthat differences in sequence would necessitate differences in cloningstrategies, for example, the use of different restriction endonucleasesmay be required to generate chimeras. Selection of existing alternativesites, or introduction via mutagenesis of alternative sites are wellestablished processes and are known to one skilled in the art.

[0492] Enzyme expression and purification can be accomplished by avariety of molecular biology methods. The examples described below teachone such method, though it is to be understood that the presentinvention is not to be limited by the method of cloning, proteinexpression, or purification. The present invention contemplates that thenucleic acid construct be capable of expression in a suitable host.Numerous methods are available for attaching various promoters and 3′sequences to a gene structure to achieve efficient expression.

[0493] 5) Site-Specific Mutagenesis

[0494] In some embodiments of the present invention, any suitabletechnique (e.g., including, but not limited to, one or more of thetechniques described above) are used to generate improved cleavageenzymes (e.g., SEQ ID NO:221) with heterologous domains. Accordingly, insome embodiments, site-specific mutagenesis (e.g., primer-directedmutagenesis using a commercially available kit such as the TransformerSite Directed mutagenesis kit (Clontech)) is used to make a plurality ofchanges thoughout a nucleic acid sequence in order to generate nucleicacid encoding a cleavage enzyme of the present invention. In someembodiments, a plurality of primer-directed mutagenesis steps arecarried out in tandem to produce a nucleic acid encoding a cleavageenzyme of the present invention.

[0495] In some embodiments, a plurality of primer directed mutagenesissteps are directed to a selected portion of a nucleic acid sequence, toproduce changes in a selected portion of a cleavage enzyme of thepresent invention. In other embodiments, a nucleic acid having changesin one selected portion is recombined with a nucleic acid havingmutations in a different selected portion (e.g., through cloning,molecular breeding, or any of the other recombination methods known inthe art), thereby creating a nucleic acid having mutations in aplurality of selected portions, and encoding a cleavage enzyme of thepresent invention. The mutations in each selected portion may beintroduced by any of the methods described above, or any combination ofsaid methods, including but not limited to methods of random mutagenesisand site-directed mutagenesis.

[0496] For example, in one illustrative embodiment of the presentinvention, the nucleic acid sequence of SEQ ID NO:222 (a nucleic acidsequence encoding the cleavage enzyme of SEQ ID NO:221) is generated bymaking a plurality of primer-directed mutations to the nucleic acidsequence of SEQ ID NO:104 (see Example 7 for the construction of SEQ IDNO:104). In some embodiments, each mutation is introduced using aseparate mutagenesis reaction. Reactions are carried out sequentiallysuch that the resulting nucleic acid (SEQ ID NO: 222) contains all ofthe mutations. In another illustrative embodiment of the presentinvention, the nucleic acid sequence of SEQ ID NO:222 is generated bymaking a plurality of primer-directed mutations, as described above, inthe nuclease portion (e.g., as diagrammed in FIG. 6) of SEQ ID NO:111.The mutant nuclease portion is then combined with the “polymerase”portion of SEQ ID NO:104 at the Not I site, using the recombinationmethods described in Example 4, thereby creating a single nucleic acidhaving SEQ ID NO:222, and encoding the cleavage enzyme of SEQ ID NO:221.Following mutagenesis, the resulting altered polypeptide is produced andtested for cleavage activity using any suitable assay (e.g., including,but not limited to, those described in Examples 1 and 6). In someembodiments, the nucleic acid sequence encoding the cleavage enzyme ofSEQ ID NO:221 (e.g., SEQ ID NO:222) is further modified using anysuitable method.

[0497] V. Reaction Design for INVADER Assay Detection of RNA Targets;

[0498] Approaches to designing INVADER assays for the detection of RNAtargets can vary depending on the needs of a particular assay. Forexample, in some embodiments, an RNA to be detected or analyzed may bepresent in a test sample at low levels, so a high level of sensitivity(i.e., a low limit of detection, or LOD) may be desirable; in otherembodiments, an RNA may abundant, and may not require an especiallysensitive assay for detection. In some embodiments, an RNA to bedetected may be similar to other RNAs in a sample that are not intendedto be detected, so that a high level of selectivity in an assay isdesirable, while in other embodiments, it may be desired that multiplesimilar RNAs be detected in a single reaction, so an assay may beprovided that is not selective with respect to the differences amongthese similar RNAs.

[0499] In some embodiments it is especially desirable to avoid detectionof any DNA molecules related to the target RNA molecules in a reaction.In some embodiments, this is accomplished by designing INVADER assayprobe sets to RNA splice junctions, such that only the properly splicedmRNAs provide the selected target sites for detection. In otherembodiments, samples are handled such that DNA remains double stranded(e.g., the nucleic acids are not heated or otherwise subjected todenaturing conditions), and is thus not available to serve as target inan INVADER assay reaction. In other embodiments, cells are lysed underconditions that leave nuclei intact, thereby containing and preventingdetection of the genomic DNA, while releasing the cytosolic mRNAs intothe lysate solution for detection by the assay.

[0500] In some embodiments, the INVADER assay is to be used fordetection or quantitation of an entire RNA having a particular variationof a sequence (e.g., a mutation a SNP, a particular spliced junction);in such embodiments, the location of the base or sequence to be detectedis a determining factor in the selection of a site for the INVADER assayprobe set to hybridize. In other embodiments, any portion of an RNAtarget may be used to indicate the presence or the amount of the entireRNA (e.g., as in gene expression analysis). In this case, the probe setsmay be directed toward a portion of the RNA selected for optimalperformance (e.g., sites determined to be particularly accessible forprobe hybridization) as a target in the INVADER assay.

[0501] The discussion of INVADER assay probe design is divided into thefollowing sections:

[0502] i. Target site selection based on accessibility

[0503] ii. Target site selection based on selectivity

[0504] iii. Oligonucleotide design

[0505] a. Target-specific regions: length and melting temperature

[0506] b. Non-complementary regions

[0507] c. Folding and dimer analysis

[0508] iv. Assay performance evaluation

[0509] v. Design and assay optimization

[0510] i. Target Site Selection Based on Accessibility

[0511] One consideration in the selection of sites for detection is theavailability of the target site for hybridization of the assay probeset. To simply use randomly selected complementary oligonucleotides fora given RNA target without prior knowledge of regions of the RNA thatallow efficient hybridization can be an ineffective approach. Forexample, it is estimated that targeting RNA with antisenseoligonucleotides based on random design results in one out of 18-20tested oligonucleotides showing significant inhibition of geneexpression (Sczakiel, Fronteirs in Biosciences 5:194 [2000]; Patzel etal, Nucleic Acids Res., 27:4328 [1999]; Peyman et al., Biol. Chem.Hoppe-Seyler 367:195 [1995]; Monia et al., Nature Med., 2:668 [1996]).Secondary and tertiary structures of RNA are thought to be the majorreasons that influence the ability of an oligonucleotide to bindtargeted regions of the RNA (Vickers et al., Nucleic Acids Res., 28:1340[2000]; Lima et al., Biochemistry 31:12055 [1992]; Uhlenbeck, J. Mol.Biol., 65:25 [1972]; Freier and Tinoco, Biochemistry 14:3310 [1975]).This is due to the hybridization kinetics and thermodynamics ofdestroying any structural motifs of the RNA and, in return, hybridizingthe complementary DNA oligonucleotide (Patzel et al., Nucleic AcidsRes., 27:4328 [1999]; Mathews et al., RNA 5:1458 [1999]). Thus, theability to identify regions of RNA that are “accessible” forhybridization is important for design and selection of effectiveoligonucleotides.

[0512] There are several experimental and theoretical methods availablefor identifying accessible regions in RNA. These include the use ofRNase-H footprinting (Ho et al., Nature Biotechnology 16:59 [1998];Mateeva et al., Nucleic Acids Res., 25:5010 [1997]; Mateeva et al.,Nature Biotechnology 16:1374 [1998]), complementary arrays ofoligonucleotide libraries (Southern et al., Nucleic Acids Res., 22:1368[1994]; Mir and Southern, Nature Biotechnology 17:788 [1999]), ribozymelibraries with random hexamer internal guide sequences (Campbell andCech, RNA 1:598 [1995]; Lan et al., Science 280:1593 [1998]), and RNAand DNA structure prediction computer programs (Sczakiel, Frontiers inBiosciences 5:194 [2000]; Patzel et al., Nucleic Acids Res., 27:4328[1999]; Zuker, Science 244:48 [1989]; Walton et al., Biotechnol.Bioeng., 65:1 [1999]). Recently, new methods have been developed thatuse primer extension to identify sites in RNAs that are accessible forhybridization. Target nucleic acids (e.g., mRNA target nucleic acids)are contacted with a plurality of primers containing a 3′ a region ofdegenerate sequence and primer extension reactions are conducted. Wherethe target nucleic acid is an RNA molecule, preferred enzymes for use inthe extension reactions are reverse transcriptases, which produce a DNAcopy of the RNA template. Folded structures present in the targetnucleic acid affect the initiation and/or efficiency of the extensionreaction. The extension products of the primers are analyzed to providea map of the accessible sites. For example, certain extension productsare not generated where the primer is complementary to a sequence thatis involved in a folded structure. Regions of the target nucleic acidthat do not allow hybridization of the primer and do not result in theproduction of an extension product are considered inaccessible sites. Incontrast, the presence of an extension product indicates that the primerwas able to bind to an accessible region of the target nucleic acid.Such methods are referred to herein as “reverse transcription withrandom oligonucleotide libraries” or “RT-ROL” (H T Allawi, et al., RNA7(2):314-27 [2001]; PCT Publication WO 01/98537, incorporated herein byreference in its entirety). The use of a physical measurement such asRT-ROL or array hybridization provides the most direct evidence of theaccessibility of a site on an RNA strand. In general, INVADER assaysdirected toward accessible regions produce stronger signals for a givenamount of RNA than assays directed toward less accessible regions of anRNA strand. For the detection of rare RNAs (e.g., fewer than about 5,000to 10,000 copies per INVADER assay reaction), or in any assay wherein itis desirable to have the best (i.e., lowest) limit of detectionpossible, it may be beneficial to start the assay design by analyzingthe RNA structure using RT-ROL or another method of physical analysis.

[0513] In other embodiments, ease of assay design may be more importantthan creation of an assay with a particularly low LOD. Structureprediction software can simplify the task of determining which parts ofan RNA are likely to be single stranded, and thus be more accessible forprobe hybridization. As first step, the sequence of an RNA to bedetected is entered into an electronic file. It may be entered manuallyor imported from a file (e.g., a sequence data file, or a wordprocessing file). In some embodiments, the sequence is downloaded from adatabase, such as GenBank or EMBL. The RNA sequence can then be analyzedusing a program such as mfold (Zucker, Science 244:48 [1989]), OligoWalk(Mathews et al., RNA 5:1458 [1999]), and variations of both (Sczakiel,Frontiers in Biosciences 5:194 [2000]; Patzel et al., Nucleic AcidsRes., 27:4328 [1999]; Walton et al., Biotechnol. Bioeng., 65:1 [1999]).

[0514] Mfold Analysis for Target RNA Structure Prediction.

[0515] The output of mfold analysis can be used in several ways toassist in identifying accessible regions of an RNA target molecule. Inone embodiment, the mfold program is used to generate an “ss count” filefor identifying regions least likely to be involved in intra-strandbaseparing. In another embodiment, the mfold program is used to generatea “.ct” file, a file used as input information for use with RNAStructure 3.5 to perform an OligoWalk analysis. In preferredembodiments, for either use, the sequence to be detected is entered intomfold. In a preferred embodiment, the settings used in the mfoldanalysis include:

[0516] Folding Temperature: 37° C. (Even though the INVADER reaction maynot be conducted at this temperature.)

[0517] % Suboptimality: 5

[0518] # foldings: 50

[0519] Window Parameter: Default

[0520] Maximum distance between paired bases: No Limit

[0521] Select BATCH folding

[0522] Enter:an e mail address where the results are to be sent whenready

[0523] Image Resolution: High

[0524] Structure Format: Bases

[0525] Base Number Frequency:Default

[0526] Structure Rotation Angle: 0

[0527] Structure Annotation: SS-Count

[0528] 1M NaCl (Australian mfold Internet site only)

[0529] When results are ready, an e mail message is sent containing theWeb address of the results. The only file that is necessary forsubsequent INVADER assay probe design analysis is the SS-Count file,which is then downloaded from the Results page. An exemplary mfoldanalysis using a GenBank entry for Human Ubiquitin (#4506712) is shownbelow:

[0530] SS-Count Analysis for Accessible Sites Identification.

[0531] The SS-Count file is then imported into an Excel spreadsheet fileand the following options are chosen: Data Type=Delimited <press Next>;Delimited=Select (x) Spaces; (x) Treat multiple delimiters as one <pressNext>; Column for data format=General. Selecting these options resultsin the import into Excel of three columns of data (FIG. 39). The firstline in the first column is the total number of stable structures mfoldfound under the parameters used in the folding. With the Ubiquitinexample, there were 12 structures found.

[0532] The rest of the first column is the RNA nucleotide positionnumbered from 1. The second column is the raw SS-Count number andrepresents the number of times the corresponding base was NOTbase-paired as part of some secondary structure. The third column is thesequence of the RNA analyzed, identifying the base at each numberedposition. By looking for bases that are involved in fewer structures(i.e., bases listed in column 3 corresponding to the higher numbers incolumn 2), it is possible to identify regions of the mRNA (identified byposition number in column 1) that are more likely to be free ofintra-strand base-pairing, and thus are more likely to be available fordetection using the INVADER assay.

[0533] One way of viewing the data is to calculate the running averageSS-count for a ten nucleotide stretch of the RNA (the Ave(10) Index) andchart the Ave(10) Index against the base pair position (See e.g., FIG.48).

[0534] An alternative plot is to graph the nucleotide position againstthe Ave(10) Index expressed as a percentage of the total number ofstructures found by mfold (FIG. 39). As with the raw SS-count table,regions of the RNA corresponding to higher numbers in the Ave(10) Indexare involved in fewer predicted folded structures. Viewing the Ave(10)Index in either a chart or graph format reduces the complexity of thedata, and can reveal longer stretches of the RNA that are more likely tobe structure-free. For example, from the graph of the running average, auser can pick out all of the major peak areas as likely regions forINVADER assay probe design. This creates an SS-Count Candidate List. Inthe Human ubiquitin example, there is one major peak and about 6 otherpeaks (FIG. 39)

[0535] The next step is to refer back to the raw (i.e., not the runningaverage) SS-Count data from within each of the peak areas, and identifythe residue where the running average is changing in magnitude, this isa local “turn” and is generally a good candidate residue to bepositioned at the INVADER assay probe set cleavage site. For example, inHuman Ubiquitin, residue Gi 19 is found at aminor local turn within aglobally accessible region (FIG. 40). The INVADER assay probe set withthe cleavage site at this location is a good performer in detection ofthis RNA. Placing the cleavage site at GI 14 did not result in betterdetection even though it had a higher %Ave(10) value. While not limitingthe present invention to any particular mechanism, and an understandingof the mechanism is not necessary to practice the invention, this islikely to be because this area was less accessible to the Probe orINVADER oligonucleotides than was position Gi 19.

[0536] OligoWalk Structure Prediction with RNA Structure 3.5 forAccessible Sites Identification

[0537] In some embodiments, the program OligoWalk, a module of thesoftware “RNAStructure” (Mathews et al., RNA 5:1458 [1999]) is used inthe selection of sites that are more likely to be accessible foroligonucleotide binding. OligoWalk uses sets of thermodynamic parametersfor both RNA and DNA, and their hybrids (Allawi and SantaLucia,Biochemistry 36:10581 [1997]; Mathews et al., J. Mol. Biol., 288:911[1999]; Sugimoto et al., Biochemistry 34:11211 [1995]) in an algorithmthat relies on mfold for RNA secondary structure prediction (Zucker,Science 244:48 [1989]). OligoWalk is designed to predict the mostfavorable regions of an RNA target for designing antisenseoligonucleotides by estimating the overall thermodynamics of hybridizingan antisense oligomer to the RNA by taking into account thethermodynamics of destroying any structural motifs in the RNA target orthe antisense oligonucleotide. The affinity of the oligomer to itstarget is expressed as an overall Gibbs free energy change of aself-structured oligomer, and of a target associating into anoligomer-target complex. This free energy is usually a negative number,indicating favorable binding, and is expressed in ‘kcal/mol’ units.OligoWalk analysis is performed with 8 to 15 base oligonucleotide sizeto resemble the average length of the analyte specific region of theSignal Probe. Plotting the total binding energy against the length ofthe RNA generates a graph of peaks and valleys. The lowest negativevalues generally indicate the most favorable sites for oligonucleotidesto bind. The most inaccessible regions have positive binding energyvalues, and generally are a poor sites for assay probe design

[0538] In a preferred embodiment, the OligoWalk module of RNA Structure3.5 is used to determine binding energies by performing an 8-baseOligoWalk using the following settings:

[0539] Break Local Structure

[0540] Include suboptimal structures

[0541] Oligo Length: 8 nt

[0542] Oligo Concentration: 100 nM

[0543] Oligo Type: DNA

[0544] Walk entire Target RNA

[0545] When these parameters have been set, the sequence file to befolded (the “.ct” output file from mfold) can be selected and opened.Once the sequence has been folded, a report can be created using theOutput menu. The report is imported into Excel and the data generatedabove is plotted. In a preferred embodiment, the OligoWalk data isgraphed with the SS-Count data. The regions displaying the lowest freeenergy values (i.e., the largest negative numbers) are generally themost likely to be accessible for hybridization. In preferredembodiments, the 3′ end and the majority of the target-binding region ofthe probe oligonucleotide complement an accessible region of the targetRNA. In particularly preferred embodiments, the majority of the bindingsite for the corresponding INVADER oligonucleotide falls within the sameaccessible region. In another preferred embodiment, the binding site foran INVADER oligonucleotide falls within a nearby accessible region.

[0546] An INVADER oligonucleotide can generally be positioned to bind toa less accessible site. While not limiting the present invention to anyparticular mechanism, it is observed that the INVADER oligonucleotidesare generally longer than probe oligonucleotides used in the INVADERassay reactions and, because they are generally designed to remain boundto the target at the reaction temperature, they will be selected to havea T_(m)s about 12 to 15° C. higher than that of a corresponding probe.Consequently, INVADER oligonucleotides may more readily break the localtarget structure, and thus may be less dependent on the accessibility ofthe target-binding site.

[0547] In selecting among accessible sites for the design of INVADERassay oligonucleotides, the base composition of the site is alsoconsidered. It has been observed that stretched of more than 4 or 5 ofthe same nucleotide in a row (e.g., . . . AAAA . . . or . . . CCCC . . .) in any portion of the binding site for the assay oligonucleotides mayreduce the performance of the probe set in the assay (e.g., byincreasing background or decreasing specificity). Thus, in preferredembodiments, any stretches comprising four or more repeated bases aregenerally avoided. Another consideration is the effect of basecomposition on lengths of the oligonucleotides in the probe set. In manycases, targeting A-T rich sequences requires the use of longeroligonucleotides for a reaction performed at a given temperature,compared to the length of oligonucleotides targeted to sequences havinga more even distribution of A-T and G-C bases. Longer oligonucleotidescan be more prone to formation of intrastrand structures and dimerstructures. Thus, it is preferred that the distribution or A-T bases andG-C bases within a target region be as close to even (i.e., about 50%G-C content) as the region to be detected permits. In particularlypreferred embodiments, the distribution of A-T and G-C positions isevenly distributed across the binding sites (e.g., not having all A-Tpositions in one half, with all G-C positions in the other).

[0548] ii. Target Site Selection Based on Selectivity

[0549] In some embodiments, probe sets are designed to examine highlyhomologous, or closely related RNA targets (i.e., targets that are verysimilar in sequence). In such embodiments, the RNA or homologous cDNAsequences are compared, e.g., using an alignment program such asMEGALIGN (DNAstar Madison, Wis.).

[0550] In some embodiments, selectivity is provided by designing probesets to detect splice junctions. Splice junctions can be identified byaligning the cDNA and gene sequences using an alignment program (e.g.MEGALIGN) or under the BLAST menu at the NCBI website (BLAST 2sequences). Splice junctions are also often listed in the GenBank report(intron/exon sites). INVADER assay oligonucleotide sets are designedsuch that the probe and INVADER oligonucleotides are complementary tothe coding strand (mRNA), generally with the cleavage site being asclose to the splice junction as possible. In some embodiments, differentsplice junctions within an mRNA are analyzed for accessibility, asdescribed above. In preferred embodiments, probe sets are designed todetect one or more splice junctions showing greater accessibilitycompared to the accessibility of other splice junctions within the sameRNA target.

[0551] In some embodiments designed to exclude detection of RNAs relatedto the target RNA, sequences are examined to identify bases that areunique to the target RNA when compared to the other similar sequencesfrom which the target is distinguished. Generally, the unique base ispositioned to hybridize to the 5′ end of the target-specific region ofthe probe oligonucleotide. In some embodiments, two adjacent bases areunique to the target compared to the related RNA. If two adjacent uniquebases are available in an appropriately accessible portion of the targetRNA, it is preferred that these bases be used as the site around whichthe probe and INVADER oligonucleotides sets are designed. In someembodiments, the two unique bases are positioned such that the site ofcleavage of the probe is between the two base-pairs they form with theprobe. In other embodiments, one of the unique bases is in the lastposition of the hybridization site of the INVADER oligonucleotide (i.e.,it is positioned to base-pair to the penultimate residue on the 3′ endof the INVADER oligonucleotide).

[0552] In some embodiments, the assay is designed to include detectionof RNAs that are similar, but not identical, to the target RNA. If theassay is being designed for inclusive detection, the compared sequencesare examined to identify sites having complete homology. Such designscan be created to detect homologous sequences in the same species orbetween species. Generally, the most homologous regions are selected ashybridization targets for probe oligonucleotides. Generally, somevariation can be tolerated, for example, if it is not at the base thatwould hybridize to the 5′ end of the target-specific region of a probe.In some embodiments, variation is accommodated by the use of degeneratebases in the INVADER assay oligonucleotides (e.g., mixtures of bases areused at positions within the synthesized probe, INVADER and/or stackeroligonucleotides, said mixtures selected to complement the mixture ofspecific bases present in the collection of related target RNAs).

[0553] iii. Oligonucleotide Design

[0554] a. Target-Specific Regions: Length and Melting Temperature

[0555] As described above in Section I (a) concerning theoligonucleotide design, in some embodiments, the length of theanalyte-specific regions are defined by the temperature selected forrunning the reaction. Starting from the desired position (e.g., avariant position or splice junction in a target RNA, or a sitecorresponding to a low free energy value in an OligoWalk analysis) aniterative procedure is used by which the length of the ASR is increasedby one base pair until a calculated optimal reaction temperature (T_(m)plus salt correction to compensate for enzyme and any other reactionconditions effects) matching the desired reaction temperature isreached. In general probes are selected to have an ASR with a calculatedT_(m) of about 60° C. if a stacking oligonucleotide is not used, and aT_(m) of about 50 to 55° C. if a stacking oligonucleotide is used (astacking oligonucleotide typically raises the T_(m) of a flanking probeoligonucleotide by about 5 to 15° C.). If the position of variation or asplice junction is a starting position, then the additions are made tothe 3′ end of the probe. Alternatively, if the 3′ end of the probe is tobe positioned at the most accessible site, the additions are in the 5′direction. In some embodiments, wherein a stacker oligonucleotide is tobe used, it is preferred that the probe be designed to have a 3′ basethat has stable stacking interaction interface with the 5′ base of thestacker oligonucleotide. The stability of coaxial stacking is highlydependent on the identity of the stacking bases. Overall, the stabilitytrend of coaxial stacking in decreasing order ispurine:purine>purine:pyrimidine≈pyrimidne:purine>pyrimidine:pyrimidine.In other embodiments employing a stacker, a less stable stackinginteraction is preferred; in such cases the probe 3′ base and/or thestacker 5′ base are selected to provide a leass stable stackinginteraction. In some embodiments, the probe 3′ base and/or the stacker5′ base are selected to have a mismatch with respect to the targetstrand, to reduce the strength of the stacking interaction.

[0556] The same principles are also followed for INVADER oligonucleotidedesign. Briefly, starting from the position N, additional residuescomplementary to the target RNA starting from residue N−1 are then addedin the upstream direction until the stability of the INVADER-targethybrid exceeds that of the probe (and therefore the planned assayreaction temperature). In preferred embodiments, the stability of theINVADER-target hybrid exceeds that of the probe by 12-15° C. In general,INVADER oligonucleotides are selected to have a T_(m) near 75° C.Software applications, such as INVADERCREATOR (Third Wave Technologies,Madison, Wis.) or Oligonucleotide 5.0 may be used to assist in suchcalculations.

[0557] If a stacking oligonucleotide is to be used, similar designprinciples are applied. The stacking oligonucleotide is generallydesigned to hybridize at the site adjacent to the 3′ end of the probeoligonucleotide, such that the stacker/target helix formed can coaxiallystack with the probe/target helix. The sequence is selected to have acalculated T_(m) of about 60 to 65° C., with the calculation based onthe use of natural bases. However, stacking oligonucleotides aregenerally synthesized using only 2′-O-methyl nucleotides, andconsequently, have actual T_(m)s that are higher than calculated byabout 0.8° C. per base, for actual T_(m)s close to 75° C.

[0558] In some embodiments, ARRESTOR oligonucleotides are included in asecondary reaction. ARRESTOR oligonucleotides are provided in asecondary reaction to sequester any remaining uncleaved probe from theprimary reaction, to preclude interactions between the primary probe andthe secondary target strand. ARRESTOR oligonucleotides are generally2′-O-methylated, and comprise a portion that is complementary toessentially all of their respective probe's target-specific region, anda portion that is complementary to at least a portion of the probe'sflap regions (e.g., six nucleotides, counted from the +1 base towardsthe 5′ end of the arm).

[0559] b. Non-Complementary Regions

[0560] Probe 5′ Arm Selection

[0561] The non-complementary arm of the probe, if present, is preferablyselected (by an iterative process as described above) to allow thesecondary reaction to be performed at a particular reaction temperature.In the secondary reaction, the secondary probe is generally cycling, andthe cleaved 5′ arm (serving as an INVADER oligonucleotide) should stablybind to the secondary target strand.

[0562] INVADER Oligonucleotide 3′ Terminal Mismatch Selection

[0563] In preferred embodiments, the 3′ base of the INVADERoligonucleotide is not complementary to the target strand, and isselected in the following order of preference (listed as INVADERoligonucleotide 3′ base/target base): C in target C/C > A/C > T/C > G/CA in target A/A > C/A > G/A > T/A G in target A/G > G/G > T/G > C/G U intarget C/U > A/U > T/U > G/U

[0564] c. Folding and Dimer Analysis

[0565] In some embodiments, the oligonucleotides proposed for use in theINVADER assay are examined for possible inter- and intra-molecularstructure formation in the absence of the target RNA. In general, it isdesirable for assay probes to have fewer predicted inter- or intramolecular interactions. In some embodiments, the program OLIGO (e.g.,OLIGO 5.0, Molecular Biology Insights, Inc., Cascade, Colo.) is used forsuch analysis. In other embodiments, the program mfold is used for theanalysis. In yet other embodiments, the RNAStructure program can be usedfor dimer analysis. The following sections provide stepwise instructionsfor the use of these programs for analysis of INVADER assayoligonucleotides.

[0566] OLIGO 5.0 Analysis for Probe Structure and InteractionPrediction.

[0567] Analysis of INVADER oligonucleotides using OLIGO 5.0 comprisesthe following steps. All menu choices are shown in UPPER CASE type.

[0568] 1. Launch OLIGO 5.0 and open a sequence file for each mRNA to beanalyzed. This is done by using a menu to select the following

[0569] Choose FILE−>NEW

[0570] Paste in longest available sequence

[0571] Choose ACCEPT & QUIT (F6)

[0572] 2. Set Program settings to default

[0573] Choose FILE−>RESET−>ORIGINAL DEFAULTS

[0574] 3. Identify Probe Oligonucleotide

[0575] Select OLIGO LENGTH to be around 16 nucleotides (open the menufor this option by using ctrl-L keystrokes).

[0576] Move the cursor indicating the 5′ end of the Current Oligo untilthe 3′ end is located at the candidate cleavage site residue.

[0577] Choose ANALYSE−>DUPLEX FORMATION−>CURRENT OLIGO (ctrl-D) for arough determination of the extent of dimer and hairpin formation.

[0578] Confirm length of analyte region corresponds with desiredreaction temperature [e.g., through the use of T_(m) calculation asdescribed in the Optimization of Reaction Conditions, I(c) of theDetailed Description of the Invention]

[0579] Select the “LOWER” button in OLIGO 5.0 to copy the anti-sensesequence (this will be the analyte-specific region of the actual probeoligonucleotide and is anti-sense to the RNA strand.)

[0580] Import into a database file.

[0581] Save to computer memory.

[0582] 4. Identify INVADER Oligonucleotide

[0583] Choose sequence adjacent to the probe oligonucleotide identifiedfrom step 3.

[0584] Select OLIGO LENGTH to 24 nucleotides

[0585] Confirm length of analyte region corresponds with desiredreaction temperature [e.g., through the use of T_(m) calculation asdescribed in the Optimization of Reaction Conditions, I(c) of theDetailed Description of the Invention, about 75° C. for INVADERoligonucleotides). Select the “LOWER” button in OLIGO 5.0 to copy thecorresponding anti-sense sequence (this will be the analyte-specificregion of the actual INVADER oligonucleotide.)

[0586] Import into a database file.

[0587] Save to computer memory.

[0588] 5. Addition of Cleaved Arm Sequence and INVADER OligonucleotideMismatch Sequence.

[0589] Export the Probe oligonucleotide as Upper Primer.

[0590] Export the INVADER oligonucleotide as Lower Primer.

[0591] EDIT UPPER PRIMER to add in a candidate arm sequence (selected,for example, as described above).

[0592] Check that the arm sequence does not create new secondarystructures (analysis performed as described above).

[0593] EDIT LOWER PRIMER to add in the 3′ mismatched nucleotide thatwill overlap into the cleavage site (selected according to theguidelines for this mismatched bases, provided above).

[0594] Select all Upper and Lower Primer boxes in the “Print/SaveOptions”

[0595] PRINT ANALYSIS of Upper (Probe) and Lower (INVADER)oligonucleotides and check for lack of stable secondary structures.

[0596] Save both mRNA sequence and oligonucleotide sequence databasefiles before quitting the program.

[0597] Generally, oligonucleotides having detected intra-molecularformations with stabilities of less than −6 ΔG are preferred. Lessstable structures represent poor substrates for CLEAVASE enzymes, andthus cleavage of such structures is less likely to contribute tobackground signal. Probe and INVADER oligonucleotides having lessaffinity for each other are more available to bind to the target,ensuring the best cycling rates.

[0598] The T_(m) of dimerized probes (i.e., probes wherein one probemolecule is hybridized to another probe molecule) should ideally belower than the T_(m) for the probe hybridized to the target, to ensurethat the probes preferentially hybridize to the target sequence at theelevated temperatures at which INVADER assay reactions are generallyconducted. Similarly, the T_(m) for the INVADER oligonucleotidehybridized either to itself or to a probe molecule should be lower thanthe INVADER oligonucleotide/target T_(m). It is preferred that dimerT_(m)s (i.e., Probe/Probe and Probe/INVADER oligonucleotide) be 25° C.or less to ensure that they will be unlikely to form at the plannedreaction temperature.

[0599] The melting temperatures for each of these complexes can bedetermined as described above in Optimization of Reaction Conditions,I(c) of the Detailed Description of the Invention, or by using the OLIGOsoftware. Once RNAs sites and several candidate INVADER assayoligonucleotide sets are selected according to the process outlinedabove, the candidate oligonucleotide sets can be ranked according to thedegree to which they comply with preferred selection rules, e.g., theirlocation on the SS-Count average plot (peak, valley, neither), and theenergetic predictions of probe and INVADER oligonucleotide interactions.In some embodiments, the ranked probe sets are tested in order of rankto identify one or more sets having suitable performance in an RNAINVADER assay. In other embodiments, several of the top ranked sets(e.g., two, three or more) are selected for testing, to rapidly identifyone or more sets having suitable or desireable performance.

[0600] Mfold Analysis for Probe Structure and Interaction Prediction

[0601] Analysis of probe and INVADER oligonucleotide interactions may beperformed using mfold for DNA provided by Michael Zuker, availablethrough Rensselaer Polytechnic Institute atbioinfo.math.rpi.edu/˜mfold/dna/forml.cgi. The analysis is performedwithout changing the default ionic conditions, and with a selectedtemperature of 37° C. and with % suboptimality set to 75. Each sequence(e.g., probe, INVADER oligonucleotide, stacker, etc.) is folded usingthe program to check for any unimolecular structure formation (e.g.,hairpins). The energies provided by mfold gives for unimolecularstructures can be used as provided, without further calculations.

[0602] Bimolecular structure formation for a given oligonucleotide isassessed by typing in the oligonucleotide sequence (5′ to 3′) followedby the sequence of a small, stable hairpin forming sequence (e.g.,CCCCCTTTTGGGGG [SEQ ID NO:707]), followed by the same oligonucleotidesequence, again listed 5′ to 3. Constraints are entered to require thatthese Ts remain single-stranded and the strings of Cs and Gs in thisspacer are basepaired. The command “F” is used to force basepairing,while the command “P” is used to prohibit basepairing, and the positionsof the forced or prohibited basepairs are counted from the 5′ end. Forexample, if the sequence of interest is a 20-mer, then the following isentered:

[0603] F 21 0 5 [this forces the C's, C21 to C25, to base pair]

[0604] P 26 0 4 [this forces the T's, T26 to T29, to be single stranded]

[0605] F 30 0 5 [this forces the G's, G30 to G34, to base pair]

[0606] On examination of the resulting structures, the stability of eachcan be estimated by subtracting the stability (i.e., the thermodynamicmeasures) of the central spacer hairpin from the total result (i.e.,Thermodynamics of possible structure=mfold structure thermodynamics−corehairpin thermodynamics). For convenience, in some embodiments, anynearest neighbor interactions between the central hairpin and dimersformed by the test sequence are ignored for this calculation; a moreaccurate analysis would require consideration of this interaction. Thecore hairpin formed by CCCCCTTTTGGGGG (SEQ ID NO:707) has the followingthermodynamics: ΔG=−5.3; ΔH=−37.8; ΔS=−104.8.

[0607] The process can be demonstrated using the following probesequence: 5′-CCCTATCTTTAAAGTTTTTAAAAAGTTTGA-3′ (SEQ ID NO:708). Theoligonucleotide sequence is examined by mfold analysis for bimolecularstructures using the following steps.

[0608] 1—In mfold sequence box type: (SEQ lD NO:137)CCCTATCTTTAAAGTTTTTAAAAAGTTTGACCCCCTTTTGGGGGCCCTATCTTTAAAGTTTTTAAAAAGTTTGA

[0609] 2—In the constraint box type:

[0610] P 36 0 4

[0611] F 31 0 5

[0612] F 40 0 5

[0613] Results (showing one):  Structure 1 dG =  −14.2      dH =  −150.5   dS =   −439.5  Tm =  69.3CCCTATCTTT  |GG         G     --------     T          AAA TTTTTAAAAA TTTGA        CCCCC T          TTT AAAAATTTTT AAATT        GGGGG T --------AG  {circumflexover ( )}G          G     TCTATCCC     T

[0614] To evaluate the stability of the duplex:CCCTATCTTT  |G          G           AAA TTTTTAAAAA TTTGA          TTT AAAAATTTTT AAATT --------AG  {circumflex over( )}G          G     TCTATCCC

[0615] the thermodyanamic values for the hairpin alone are subtractedfrom the values for the complete structure:

[0616] ΔG=−14.2−(−5.3)=−8.9,

[0617] ΔH=−150.5−(−37.8)=−112.7,

[0618] ΔS=−439.5−(−104.8)=−334.7,

[0619] Using a calculation wherein T_(m) (° C.)={ΔH/[ΔS+Rln(CT/4)]}−273.15, wherein R is the gas constant 1.987 (cal/K.mol), Inis the natural log, and CT is the total single strand concentration inMolar, this results in a calculated T_(m) of 46.1° C. for thenon-hairpin portion of the structure.

[0620] The above method is not limited to the use of the core hairpinsequence CCCCCTTTTGGGGG but rather any stable hairpin sequences can beused. For example, CGCGCGGAACGCGCG (SEQ ID NO:138) or CCCGGGTTTTCCCGGG(SEQ ID NO:139). However, if a different hairpin sequence is used, oneneeds to calculate its stability using mfold and use its thermodynamicsin the subsequent calculations.

[0621] RNAStructure for Oligonucleotide Interaction Prediction

[0622] Dimer formation can also be evaluated using the RNAStructureprogram. Unlike mfold, RNAStructure allows the calculation of allpossible oligonucleotide-oligonucleotide interactions and provides anoutput .ct file. One can then view the structures using any .ct viewingprogram such as RNAStructure or RNAvis (1997, P. Rijk, University ofAntwerp (UIA), available on the Internet at rrna.uia.ac.be/rnavis) andevaluate the stability of any dimer formation using the nearest-neighbormodel (Borer et al., 1974) and DNA nearest-neighbor parameters (Allawi &SantaLucia, 1997).

[0623] For example, to evaluate the propensity of the sequence 5′AGGCGCACCAATTTGGTGTT 3′ (SEQ ID NO:140) for dimer formation using theDNA Fold Intermolecular module of RNAStructure, the sequence is savedinto a file (e.g., probe.seq) and the following parameters are set:

[0624] Sequence file 1: probe.seq

[0625] Sequence file 2: probe.seq

[0626] CT file: dimer.ct

[0627] Max % Energy difference: 50

[0628] Max number of structures: 20

[0629] Window size: do not change

[0630] After the calculation is done, one can view the resulting .ctfile using the “view” module of RNAStructure. Generally, there will beseveral structures within the .ct file. The view module is used to viewthem individually. One of the dimers that the test sequece, above, canform according to RNAStructure is: AGGCG             TT     CACCAATTTGGTG      GTGGTTTAACCAC    TT             GCGGA

[0631] According to the nearest-neighbor model (i.e., using DNAnearest-neighbor and mismatch parameters [Allawi & SantaLucia, 1997]),the stability of this duplex in 1M NaCl and at a probe concentration of100 μM is:

[0632] ΔG°₃₇=−10.07

[0633] ΔH=−87.6

[0634] ΔS=−250.1

[0635] Tm=50.1° C.

[0636] By changing the identities of Sequence Files 1 & 2, RNAStructurecan be used to evaluate the possibility of any dimer formation betweenpairs of all of the DNA oligonucleotides present in an INVADER assayreaction.

[0637] iv. Assay Performance Evaluation

[0638] Probe sets selected according to the guidelines provided abovecan be tested in the INVADER assay to evaluate performance. While theoligonucleotides are designed to perform at or near a particular desiredreaction temperature, the best performance for a given design may not beprecisely at the intended temperature. Thus, in evaluating any newINVADER assay probe set, it can be helpful to examine the performance inthe INVADER assay conducted at several different reaction temperatures,over a range of about 10 to 15° C., centered around the designedtemperature. For convenience, temperature optimization can be performedon a temperature gradient thermocycler with a fixed amount of RNA (e.g.,2.5 amoles of an in vitro transcript per reaction), and for a fixedamount of time (e.g., 1 hour each for Primary and Secondary reactions).The temperature gradient test will reveal the temperature at which thedesigned probe set produces the best performance (e.g., the highestlevel of target-specific signal compared to background signal, generallyexpressed as a multiple of the zero-target background signal, or “foldover zero”).

[0639] The results can be examined to see how close the measuredtemperature optimum is to the intended temperature of operation. In someembodiments, it is desirable to have probe sets that operate at or neara pre-selected temperature. If the measured temperature optimum ishigher than the desired reaction temperature, a probe design can bealtered in ways that tend to reduce the probe/target T_(m) (e.g.,shortened by one or more bases, or altered to contain one or moremismatched bases). In some embodiments, wherein a stackeroligonucleotide is not used, wherein the reaction temperature is morethan 7° C. above the desired reaction temperature, and wherein theperformance (e.g., the fold over zero) is acceptable, use of a 3′mismatch on the probe oligonucleotide is likely to lower the reactiontemperature without otherwise altering the assay performance.

[0640] An LOD determination can be made by performing reactions onvarying amounts of target RNA (e.g., an in vitro transcript control RNAof known concentration). In preferred embodiments, a designed assay hasan LOD of less than 0.05 attomole. In particularly preferredembodiments, a designed assay has an LOD of less than 0.01 attomole. Itis contemplated that the same guideline provided above for reducing theLOD of a designed assay may be used for the purpose of raising the LODof a designed assay, i.e., to make it LESS sensitive to the presence ofa target RNA. For example, it may be desirable to detect an abundant RNAand a rare RNA in the same reaction. In such a reaction, it may bedesirable to attenuate the signal generated for the abundant RNA so thatit does not overwhelm the signal from the rarer species. In someembodiments this may be done by designing probe sets for reduced signalgeneration, e.g., an LOD of at least (not less than) 0.5 attomoles. Insome embodiments, a single step INVADER assay may be used for detectionof abundant targets in a sample, while sequential INVADER reactions toamplify signal, as described in Section II, may be used for lessabundant analytes in the same sample. In preferred embodiments, thesingle step and the sequential INVADER assay reactions for the differentanalytes are performed in a single reaction.

[0641] In some embodiments, time course reactions are run, wherein theaccumulation of signal for a known amount of target is measured forreactions run for different lengths of time. This measurement willestablish the linear ranges, i.e., the ranges in which accuratequantitative measurements can be made using a given assay design, withrespect to time and starting target RNA level.

[0642] V. Design and Assay Optimization

[0643] Some designed assays may not meet the preferred performancecriteria described above. A number of variations on the performance ofINVADER assay reactions have been described herein. In optimizingperformance of the INVADER assay for the detection of RNA targets, thesevariations may be used alone or in combination. For example, in someembodiments, a stacker oligonucleotide is employed. While not limitingthe present invention to any particular mechanism of action, in someembodiments, a stacker oligonucleotide may enhance performance of anassay by altering the hybridization characteristics (e.g., T_(m)) of aprobe or an INVADER oligonucleotide. In some embodiments, a stackeroligonucleotide may increase performance by enabling the use of ashorter probe. In other embodiments, a stacker oligonucleotide mayenhance performance by altering the folded structure of the targetnucleic acid. In yet other embodiments, the enhancing activity of thestacker oligonucleotide may involve these and other mechanisms incombination.

[0644] In other embodiments, the target site may be shifted. In someembodiments, reactions are optimized by testing multiple probe sets thatshift along a suspected accessible site. In preferred embodiments, suchprobe sets shift along the accessible site in one to two baseincrements. In embodiments wherein accessible sites have previously beenpredicted only by computer analysis, physical detection of theaccessible sites may be employed to optimize a probe set design. Inpreferred embodiments, the RT-ROL method of detecting accessible sitesis employed. In some embodiments, optimization of a probe set design mayrequire shifting of the target site to a newly identified accessiblesite.

[0645] In some embodiments, e.g., wherein an accessible site has beenidentified yet probe set performance is low, a change in the design of aprobe 5′ arm may improve assay performance without altering the sitetargeted. In other embodiments, altering the length of an ARRESTORoligonucleotide (e.g., increasing the length of the portion that iscomplementary to the 5′ arm region of the probe) may reduce backgroundsignal, thus increasing the probe stet performance.

[0646] Other variations on oligonucleotide design may be employed toalter performance in an assay. Some modifications may be employed toshift the ideal operating temperature of a probe set design into apreferred temperature range. For example, the use of shorteroligonucleotides and the incorporation of mismatches generally act toreduce the T_(m)s, and thus reduce the ideal operating temperatures, ofdesigned oligonucleotides. Conversely, the use of longeroligonucleotides and the employment of stacking oligonucleotidesgenerally act to increase the T_(m)s, and thus increase the idealoperating temperatures of the designed oligonucleotides.

[0647] Other modifications may be employed to alter other aspects ofoligonucleotide performance in an assay. For example, the use of baseanalogs or modified bases can alter enzyme recognition of theoligonucleotide. In some embodiments, such modified bases are used toprotect a region of an oligonucleotide from nuclease cleavage. In otherembodiments, modified bases are used to affect the ability of anoligonucleotide to participate as a member of a cleavage structure thatis not in a position to be cleaved (e.g., to serve as an INVADERoligonucleotide to enable cleavage of a probe). These modified bases maybe referred to as “blocker” or “blocking” modifications. In someembodiments, assay oligonucleotides incorporate 2′-O-methylmodifications. In other embodiments, assay oligonucleotides incorporate3′ terminal modifications (e.g., NH2, 3′ hexanol, 3′ phosphate, 3′biotin).

[0648] In yet other embodiments, the components of the reaction may bealtered to affect assay performance. For example, oligonucleotideconcentrations may be varied. Oligonucleotide concentrations can affectmultiple aspects of the reaction. Since melting temperatures ofcomplexes are partly a function of the concentrations of the componentsof the complex, variation of the concentrations of the oligonucleotidecomponents can be used as one facet of reaction optimization. In themethods of the present invention, ARRESTOR oligonucleotides may be usedto modulate the availability of the primary probe oligonucleotides in anINVADER assay reaction. In some embodiments, an ARRESTOR oligonucleotidemay be excluded. Other reaction components may also be varied, includingenzyme concentration, salt and divalent ion concentration and identity.

[0649] VI. Kits for Performing the RNA INVADER Assay

[0650] In some embodiments, the present invention provides kitscomprising one or more of the components necessary for practicing thepresent invention. For example, the present invention provides kits forstoring or delivering the enzymes of the present invention and/or thereaction components necessary to practice a cleavage assay (e.g., theINVADER assay). The kit may include any and all components necessary ordesired for the enzymes or assays including, but not limited to, thereagents themselves, buffers, control reagents (e.g., tissue samples,positive and negative control target oligonucleotides, etc.), solidsupports, labels, written and/or pictorial instructions and productinformation, inhibitors, labeling and/or detection reagents, packageenvironmental controls (e.g., ice, desiccants, etc.), and the like. Insome embodiments, the kits provide a sub-set of the required components,wherein it is expected that the user will supply the remainingcomponents. In some embodiments, the kits comprise two or more separatecontainers wherein each container houses a subset of the components tobe delivered. For example, a first container (e.g., box) may contain anenzyme (e.g., structure specific cleavage enzyme in a suitable storagebuffer and container), while a second box may contain oligonucleotides(e.g., INVADER oligonucleotides, probe oligonucleotides, control targetoligonucleotides, etc.). In some embodiments one or more the reactioncomponents may be provided in a predispensed format (i.e., premeasuredfor use in a step of the procedure without re-measurement orre-dispensing). In some embodiments, selected reaction components aremixed and predispensed together. In preferred embodiments, predispensedreaction components are predispensed and are provided in a reactionvessel (including but not limited to a reaction tube or a well, as in,e.g., a microtiter plate). In particularly preferred embodiments,predispensed reaction components are dried down (e.g., desiccated orlyophilized) in a reaction vessel.

[0651] Additionally, in some embodiments, the present invention providesmethods of delivering kits or reagents to customers for use in themethods of the present invention. The methods of the present inventionare not limited to a particular group of customers. Indeed, the methodsof the present invention find use in the providing of kits or reagentsto customers in many sectors of the biological and medical community,including, but not limited to customers in academic research labs,customers in the biotechnology and medical industries, and customers ingovernmental labs. The methods of the present invention provide for allaspects of providing the kits or reagents to the customers, including,but not limited to, marketing, sales, delivery, and technical support.

[0652] In some embodiments of the present invention, quality control(QC) and/or quality assurance (QA) experiments are conducted prior todelivery of the kits or reagents to customers. Such QC and QA techniquestypically involve testing the reagents in experiments similar to theintended commercial uses (e.g., using assays similar to those describedherein). Testing may include experiments to determine shelf life ofproducts and their ability to withstand a wide range of solution and/orreaction conditions (e.g., temperature, pH, light, etc.).

[0653] In some embodiments of the present invention, the compositionsand/or methods of the present invention are disclosed and/ordemonstrated to customers prior to sale (e.g., through printed orweb-based advertising, demonstrations, etc.) indicating the use orfunctionality of the present invention or components of the presentinvention. However, in some embodiments, customers are not informed ofthe presence or use of one or more components in the product being sold.In such embodiments, sales are developed, for example, through theimproved and/or desired function of the product (e.g., kit) rather thanthrough knowledge of why or how it works (i.e., the user need not knowthe components of kits or reaction mixtures). Thus, the presentinvention contemplates making kits, reagents, or assays available tousers, whether or not the user has knowledge of the components orworkings of the system.

[0654] Accordingly, in some embodiments, sales and marketing effortspresent information about the novel and/or improved properties of themethods and compositions of the present invention. In other embodiments,such mechanistic information is withheld from marketing materials. Insome embodiments, customers are surveyed to obtain information about thetype of assay components or delivery systems that most suits theirneeds. Such information is useful in the design of the components of thekit and the design of marketing efforts.

[0655] VII. The INVADER Assay for Direct Detection and Measurement ofSpecific RNA Analytes.

[0656] The following section provides a few illustrative examples ofmRNAs that may be detected or measured using the methods, compositionsand systems of the present invention. Application of the methods andcompositions of the present invention is not limited to these targetnucleic acid.

[0657] Housekeeping Controls

[0658] RNAs that are generally present in predicable or invariantamounts in test samples provide useful control targets for detectionassays. These controls can be useful in several ways, including but notlimited to providing confirmation of the proper function of an assay,and as a standard against which a test result for another RNA can becompared or measured to aid in interpretation of a result. mRNAs for thefollowing genes find particular use in the methods of the presentinvention.

[0659] Human Ubiquitin and Mouse/Rat Ubiquitin

[0660] The ubiquitin system is a major pathway for selective proteindegradation. Degradation by this system is instrumental in a variety ofcellular functions such as DNA repair, cell cycle progression, signaltransduction, transcription, and antigen presentation. The ubiquitinpathway also eliminates proteins that are misfolded, misplaced, or thatare in other ways abnormal. This pathway requires the covalentattachment of ubiquitin (E1), a highly conserved 76 amino acid protein,to defined lysine residues of substrate proteins.

[0661] Human, Rat and Mouse glyceraldehyde-3-phosphate Dehydrogenase(GAPDH)

[0662] GAPDH is an important enzyme in the glycolysis andgluconeogenesis pathways. This homotetrameric enzyme catalyzes theoxidative phosphorylation of D-glyceraldehyde-3-phosphate to1,3-diphosphoglycerate in the presence of cofactor and inorganicphosphate. A variety of diverse biological properties of GAPDH have beenreported. These include functions in endocytosis, mRNA regulation, tRNAexport, DNA replication, DNA repair, and neuronal apoptosis.

[0663] Cytokines

[0664] A growing family of regulatory proteins that deliver signalsbetween cells of the immune system has been identified. Calledcytokines, these proteins have been found to control the growth anddevelopment, and bioactivities, of cells of the hematopoietic and immunesystems. Cytokines exhibit a wide range of biological activities withtarget cells from bone marrow, peripheral blood, fetal liver, and otherlymphoid or hematopoietic organs. The present invention describesmethods for the detection of expression of cytokines, including but notlimited to of the exemplary members of the cytokine family listed below.

[0665] Human Oncostatin M

[0666] Oncostatin M is a secreted single-chain polypeptide cytokine thatregulates the growth of certain tumor-derived and normal cell lines. Anumber of cell types have been found to bind the oncostatin M protein.While it has been shown to inhibit proliferation of a number of tumorcell types, it has also been implicated in stimulating proliferation ofKaposi's sarcoma cells.

[0667] Human Transforming Growth Factor-Beta (TGF-β)

[0668] Transforming growth factor-beta (TGF-beta) is a member of afamily of structurally-related cytokines that elicit a variety ofresponses, including growth, differentiation, and morphogenesis, in manydifferent cell types. In vertebrates, at least five different forms ofTGF-beta, termed TGF-beta1 to TGF-beta5, have been identified; they allshare a high degree (60%-80%) of amino-acid sequence identity. WhileTGF-beta1 was initially characterized by its ability to induceanchorage-independent growth of normal rat kidney cells, its effects onmost cell types are anti-mitogenic. It is strongly growth-inhibitory formany types of cells, including both normal and transformed epithelial,endothelial, fibroblast, neuronal, lymphoid, and hematopoietic cells. Inaddition, TGF-beta plays a central role in regulating the formation ofextracellular matrix and cell-matrix adhesion processes.

[0669] Human Monocyte Chemoattractant Protein-1 (MCP-1)

[0670] Within this family of cytokines, an emerging group of chemotacticcytokines, also called chemokines or intercrines, has been identified.Two subfamilies of chemokines have been recognized, alpha and beta,based on chromosomal location and the arrangement of the cysteineresidues.

[0671] The human genes encoding the beta subfamily proteins are locatedon chromosome 17 (their mouse counterparts are clustered on mousechromosome 11, which is the counterpart of human chromosome 17).Homology in the beta subfamily ranges from 28-45% intraspecies, from25-55% interspecies. An exemplary member is the human protein MCP-1(monocyte chemoattractant protein-1). MCP-1 exerts several effectsspecifically on monocytes. It is a potent chemoattractant for humanmonocytes in vitro and can stimulate an increase in cytosolic freecalcium and the respiratory burst in monocytes. MCP-1 has been reportedto activate monocyte-mediated tumoristatic activity, as well as toinduce tumoricidal activity. MCP-1 has been implicated as an importantfactor in mediating monocytic infiltration of tissues inflammatoryprocesses such as rheumatoid arthritis and alveolitis. The factor mayalso play a fundamental role in the recruitment of monocyte-macrophagesinto developing atherosclerotic lesions.

[0672] Human Tumor Necrosis Factor Alpha (TNF-α)

[0673] Tumor necrosis factor alpha (TNF-alpha also cachectin) is animportant cytokine that plays a role in host defense. The cytokine isproduced primarily in macrophages and monocytes in response toinfection, invasion, injury, or inflammation. Some examples of inducersof TNF-alpha include bacterial endotoxins, bacteria, viruses,lipopolysaccharide (LPS) and cytokines including GM-CSF, IL-1, IL-2 andIFN-gamma.

[0674] Despite the protective effects of the cytokine, overexpression ofTNF-alpha often results in disease states, particularly in infectious,inflammatory and autoimmune diseases. This process may involve theapoptotic pathways. High levels of plasma TNF-alpha have been found ininfectious diseases such as sepsis syndrome, bacterial meningitis,cerebral malaria, and AIDS; autoimmune diseases such as rheumatoidarthritis, inflammatory bowel disease (including Crohn's disease),sarcoidosis, multiple sclerosis, Kawasaki syndrome, graft-versus-hostdisease and transplant (allograft) rejection; and organ failureconditions such as adult respiratory distress syndrome, congestive heartfailure, acute liver failure and myocardial infarction. Other diseasesin which TNF-alpha is involved include asthma, brain injury followingischemia, non-insulin-dependent diabetes mellitus, insulin-dependentdiabetes mellitus, hepatitis, atopic dermatitis, and pancreatitis.Further, inhibitors of TNF-alpha have been suggested to be useful forcancer prevention. Elevated TNF-alpha expression may also play a role inobesity. TNF-alpha was found to be expressed in human adipocytes andincreased expression, in general, correlated with obesity.

[0675] Human Interleukin-6 (IL-6)

[0676] IL-6 is the standardized name of a cytokine called B lymphocytedifferentiating factor, interferon beta2, 26 K_(d) protein,hybridoma/plasmacytoma growth factor, hepatocyte stimulating factor,etc.

[0677] IL-6 induces activated B cells to be differentiated into antibodyforming cells. For T cells, IL-6 induces T cells stimulated by mitogensto produce IL-2 and induces the expression of IL-2 receptor on a certainT cell line or thymocytes. For blood forming cells, IL-6 induces thegrowth of blood forming stem cells synergistically in the presence ofIL-3. Furthermore, recently, it was reported that 1L-6 acted likethrombopoietin.

[0678] IL-6 is produced by various cells. It is produced by lymphocytesand is also produced by human fibroblasts stimulated by Poly (I)-Poly(C) and cycloheximide. Murine IL-6 is produced in mouse cells, which arestimulated by Poly (A)-Poly (U). Inducers for stimulation are diverse,and include known cytokines such as IL-1, TNF and IFN-beta, growthfactors such as PDGF and TGF-beta, LPS, PMA, PHA, cholera toxin, etc.Moreover, it is reported that human vascular endothelial cells,macrophages, human glioblastomas, etc. also produce IL-6. Furthermore,it is also known that the productivity can be further enhanced bystimulating cells using an inducer and subsequently treating the cellsby a metabolic inhibitor such as verapamil, cycloheximide or actinomycinD, etc.

[0679] Human Interleukin 1beta (IL-1>)

[0680] Interleukin-1 (IL-1) is important to the activation of T and Blymphocytes and mediates many inflammatory processes. Two distinct formsof IL-1 have been isolated and expressed; termed IL-1beta and IL-1alpha.IL-1beta is the predominant form produced by human monocytes both at themRNA and protein level. The two forms of human IL-1 share only 26% aminoacid homology. Despite their distinct polypeptide sequences, the twoforms of IL-1 have structural similarities, in that the amino acidhomology is confined to discrete regions of the IL-1 molecule. The twoforms of IL-1 also possess identical biological properties, includinginduction of fever, slow wave sleep, and neutrophilia, T- andB-lymphocyte activation, fibroblast proliferation, cytotoxicity forcertain cells, induction of collagenases, synthesis of hepatic acutephase proteins, and increased production of colony stimulating factorsand collagen. IL-1 also activates endothelial cells, resulting inincreased leukocyte adhesiveness, PGI₂ and PGE₂ (prostaglandins)release, and synthesis of platelet activating factor, procoagulantactivity, and a plasminogen activator inhibitor. Clearly, IL-1 plays acentral role in local and systemic host responses. Because many of thebiological effects of IL-1 are produced at picomolar (pg) concentrationsin vivo, IL-1 production is likely a fundamental characteristic of hostdefense mechanisms.

[0681] Human Interleukin 2 (IL-2)

[0682] Interleukin-2 (IL-2) is the main growth factor of T lymphocytes.By regulating T helper lymphocyte activity IL-2 increases the humoraland cellular immune responses. By stimulating cytotoxic CD8 T cells andNK cells, this cytokine participates in the defense mechanisms againsttumors and viral infections. IL-2 is used in therapy against metastaticmelanoma and renal adenocarcinoma. IL-2 is used in clinical trials inmany forms of cancer. It is also used in HIV infected patients and leadsto a significant increase in CD4 counts. Human IL-2 is a protein of 133amino acids (aa) composed of four alpha helices connected by loops ofvarious length, its tridimensional structure has been established. IL-2Ris composed of three chains alpha, beta and gamma. IL2Ralpha controlsthe affinity of the receptor IL-2Rbeta and IL-2Rgamma are responsiblefor IL-2 signal transduction. The different molecular areas of IL-2interacting with the three chains of the IL-2 R have been defined. Morespecifically it has been determined that a helix A as well as the NH2terminal area of IL-2 (residues 1 to 30) control the interactionsIL-2/IL-2Rbeta.

[0683] Human Interleukin 8 (IL-8)

[0684] Human IL-8 is a cytokine that has variously been calledneutrophil-activating protein, neutrophil chemotactic factor (NCF) andT-cell chemotactic factor. IL-8 can be secreted by several types ofcells upon appropriate stimulation. IL-8 is secreted by activatedmonocytes and macrophages as well as by embryonic fibroblasts.

[0685] IL-8 is known to induce neutrophil migration and to activatefunctions of neutrophils such as degranulation, release of superoxideanion and adhesion to the endothelial cell monolayer. There are a numberof conditions that are known to involve leukocyte infiltration intolesions. These include puhnonary diseases such as pulmonary cysticfibrosis, idiopathic pulmonary fibrosis, adult respiratory distresssyndrome, sarcoidosis and empyema; dermal diseases such as psoriasis,rheumatoid arthritis; and inflammatory bowel disease (Crohn's Disease).

[0686] The amino acid sequence characterizing human IL-8 was describedby Matsushima, et al. in PCT application WO89/08665. More recently, itwas shown that monocyte-derived IL-8 was evidently variably processed atthe N-terminus and that the IL-8 originally disclosed by Matsushima etal. was accompanied by two forms of the factor which had seven or fiveadditional amino acids at the N-terminus (Yoshimura, et al., Mol Immunol26:87 [1989]). The longest form accounted for about 8%, the next longestform for about 47%, and the shortest form for about 45% of the totalIL-8 derived from monocytes.

[0687] Human Interleukin 10 (IL-10)

[0688] Interleukin-10 (IL-10), a recently discovered lymphokine, wasoriginally described as an inhibitor of interferon-gamma synthesis andis postulated as a major mediator of the humoral class of immuneresponse. Two classes of often mutually exclusive immune responses arethe humoral (antibody-mediated) and the delayed-type hypersensitivity.

[0689] It is postulated that these two differing immune responses mayarise from two types of helper T-cell clones, namely Th1 and Th2 helperT-cells, which demonstrate distinct cytokine secretion patterns. MouseTh1 cell clones secrete interferon-gamma, and IL-2 and preferentiallyinduce the delayed-type hypersensitivity response while Th-2 cell clonessecrete IL-4, IL-5 and IL-10 and provide support for the humoralresponses. The contrast in immune response could result becauseinterferon-gamma secreted by the Th1 cell clones inhibits Th2 cloneproliferation in vitro, while IL-10 secreted by the Th2 cell clonesinhibits cytokine secretion by the Th1 cell clones. Thus the twoT-helper cell types may be mutually inhibitory and may provide theunderpinning for the two dissimilar immune responses.

[0690] IL-10 has been cloned and sequenced from both murine and human Tcells. Both sequences contain an open reading frame encoding apolypeptide of 178 amino acids with an N-terminal hydrophobic leadersequence of 18 amino acids, and have an amino acid sequence homology of73%.

[0691] Human Interleukin 4 (IL-4)

[0692] Interleukin-4 (IL-4, also known as B cell stimulating factor, orBSF-1) was originally characterized by its ability to stimulate theproliferation of B cells in response to low concentrations of antibodiesdirected to surface immunoglobulin. More recently, IL-4 has been shownto possess a far broader spectrum of biological activities, includinggrowth co-stimulation of T cells, mast cells, granulocytes,megakaryocytes, and erythrocytes. In addition, IL-4 stimulates theproliferation of several IL-2- and IL-3-dependent cell lines, inducesthe expression of class II major histocompatibility complex molecules onresting B cells, and enhances the secretion of IgE and IgG1 isotypes bystimulated B cells. Both murine and human IL-4 have been definitivelycharacterized by recombinant DNA technology and by purification tohomogeneity of the natural murine protein.

[0693] The biological activities of IL-4 are mediated by specific cellsurface receptors for IL-4 that are expressed on primary cells and invitro cell lines of mammalian origin. IL-4 binds to the receptor, whichthen transduces a biological signal to various immune effector cells.

[0694] Human Interferon Gamma (IFN-γ)

[0695] Like the interleukins, interferons belong to the class of thecytokines and are listed in various classes: interferon-alpha,interferon-beta, interferon-gamma, interferon-omega and interferon-tau.Interferon-gamma is a glycoprotein, the amino acid sequence of which hasbeen known since 1982. In the mature condition the interferon-gamma has143 amino acids and a molecular weight of 63 to 73 kilodaltons.

[0696] The tertiary and quaternary structure of the non-glycosylisedprotein was clarified in 1991. According to this, interferon-gammaexists as a homodimer, the monomers being orientated in contrarydirections in such a way that the C-terminal end of one monomer islocated in the vicinity of the N-terminal end of the other monomer. Eachof these monomers in all has six alpha-helices. Interferon-gamma is alsocalled immunointerferon, as it has non-specific antiviral,antiproliferative and in particular immunomodulatory effects. Itsproduction in T-helper-lymphocytes is stimulated by mitogens andantigens. The effect of the expressed interferon-gamma has not yet beenprecisely clarified, but is subject to intensive research. Inparticular, interferon-gamma leads to the activation of macrophages andto the synthesis of histocompatability antigens of the class 2. Invitro, the activity of interferon-gamma is normally determined as areduction in the virus-induced cytopathic effect, which arises fromtreatment with interferon-gamma. Due to its antigen-non-specificantiviral, antiproliferative and immunomodulatory activity it issuitable as a human therapeutic agent, for example of kidney tumours andchronic granulomatosis.

[0697] Cytochrome P450s

[0698] The term cytochrome P-450 refers to a family of enzymes (locatedon the endoplasmic reticulum, with high concentrations of the proteinsin the cells of the liver and small intestine) that are primarilyresponsible for the metabolism of xenobiotics such as drugs, carcinogensand environmental chemicals, as well as several classes of endobioticssuch as steroids and prostaglandins. Members of the cytochrome P450family are present in varying levels and their expression and activitiesare controlled by variables such as chemical environment, sex,developmental stage, nutrition and age.

[0699] More than 200 cytochrome P450 genes have been identified. Thereare multiple forms of these P450 genes and each of the individual formsexhibit degrees of specificity towards individual chemicals in the aboveclasses of compounds. In some cases, a substrate, whether it be drug orcarcinogen, is metabolized by more then one of the cytochromes P450.Genetic polymorphisms of cytochromes P450 result inphenotypically-distinct subpopulations that differ in their ability toperform biotransformations of particular drugs and other chemicalcompounds.

[0700] The present invention provides methods for the detectioncytochrome P450 mRNAs, including but not limited to, Human CYP 1A1,Human CYP 1A2, Human CYP 2B1, Human CYP 2B2, Human CYP 2B6, Human CYP2Cl9, Human CYP 2C9, Human CYP 2D6, Human CYP 3A4, Human CYP 3A5, HumanCYP 3A7, Rat CYP 2E1, Rat CYP 3A1, Rat CYP 3A2, Rat CYP 4A1, Rat CYP 4A2and Rat CYP 4A3.

EXAMPLES

[0701] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0702] In the disclosure which follows, the following abbreviationsapply: Afu (Archaeoglobus fulgidus); Mth (Methanobacteriumthermoautotrophicum); Mja (Methanococcus jannaschii); Pfu (Pyrococcusfuriosus); Pwo (Pyrococcus woesei); Taq (Thermus aquaticus); Taq DNAP,DNAPTaq, and Taq Pol I (T aquaticus DNA polymerase I); DNAPStf (theStoffel fragment of DNAPTaq); DNAPEc1 (E. coli DNA polymerase I); Tth(Thermus thermophilus); Ex. (Example); Fig. (Figure); ° C. (degreesCentigrade); g (gravitational field); hr (hour); min (minute); olio(oligonucleotide); rxn (reaction); vol (volume); w/v (weight to volume);v/v (volume to volume); BSA (bovine serum albumin); CTAB(cetyltrimethylammonium bromide); HPLC (high pressure liquidchromatography); DNA (deoxyribonucleic acid); p (plasmid); μl(microliters); ml (milliliters); μg (micrograms); mg (milligrams); M(molar); mM (milliMolar); μM (microMolar); pmoles (picomoles); amoles(attomoles); zmoles (zeptomoles); nm (nanometers); kdal (kilodaltons);OD (optical density); EDTA (ethylene diamine tetra-acetic acid); FITC(fluorescein isothiocyanate); SDS (sodium dodecyl sulfate); NaPO₄(sodium phosphate); NP-40 (Nonidet P-40); Tris(tris(hydroxymethyl)-aminomethane); PMSF (phenylmethylsulfonylfluoride);TBE (Tris-Borate-EDTA, i.e., Tris buffer titrated with boric acid ratherthan HCl and containing EDTA); PBS (phosphate buffered saline); PPBS(phosphate buffered saline containing 1 mM PMSF); PAGE (polyacrylamidegel electrophoresis); Tween (polyoxyethylene-sorbitan); ATCC (AmericanType Culture Collection, Rockville, Md.); Coriell (Coriell CellRepositories, Camden, N.J.); DSMZ (Deutsche Sammlung von Mikroorganismenund Zellculturen, Braunschweig, Germany); Ambion (Ambion, Inc., Austin,Tex.); Boehringer (Boehringer Mannheim Biochemical, Indianapolis, Ind.);MJ Research (MJ Research, Watertown, Mass.; Sigma (Sigma ChemicalCompany, St. Louis, Mo.); Dynal (Dynal A. S., Oslo, Norway); Gull (GullLaboratories, Salt Lake City, Utah); Epicentre (Epicentre Technologies,Madison, Wis.); Lampire (Biological Labs., Inc., Coopersberg, Pa.); MJResearch (MJ Research, Watertown, Mass.); National Biosciences (NationalBiosciences, Plymouth, Minn.); NEB (New England Biolabs, Beverly,Mass.); Novagen (Novagen, Inc., Madison, Wis.); Promega (Promega, Corp.,Madison, Wis.); Stratagene (Stratagene Cloning Systems, La Jolla,Calif.); Clonetech (Clonetech, Palo Alto, Calif.) Pharmacia (Pharmacia,Piscataway, N.J.); Milton Roy (Milton Roy, Rochester, N.Y.); Amersham(Amersham International, Chicago, Ill.); and USB (U.S. Biochemical,Cleveland, Ohio). Glen Research (Glen Research, Sterling, Va.); Coriell(Coriell Cell Repositories, Camden, N.J.); Gentra (Gentra, Minneapolis,Minn.); Third Wave Technologies (Third Wave Technologies, Madison,Wis.); PerSeptive Biosystems (PerSeptive Biosystems, Framington, Mass.);Microsoft (Microsoft, Redmond, Wash.); Qiagen (Qiagen, Valencia,Calif.); Molecular Probes (Molecular Probes, Eugene, Oreg.); VWR (VWRScientific,); Advanced Biotechnologies (Advanced Biotechnologies, INC.,Columbia, Md.); and Perkin Elmer (also known as PE Biosytems and AppliedBiosystems, Foster City, Calif.).

Example 1 Rapid Screening of Colonies for 5′ Nuclease Activity

[0703] The native 5′ nucleases and the enzymes of the present inventioncan be tested directly for a variety of functions. These include, butare not limited to, 5′ nuclease activity on RNA or DNA targets andbackground specificity using alternative substrates representingstructures that may be present in a target detection reaction. Examplesof nucleic acid molecules having suitable test structures are shownschematically in FIGS. 18A-D and FIGS. 21-24. The screening techniquesdescribed below were developed to quickly and efficiently characterize5′ nucleases and to determine whether the new 5′ nucleases have anyimproved or desired activities. Enzymes that show improved cycling rateson RNA or DNA targets, or that result in reduced target-independentcleavage merit more thorough investigation. In general, the modifiedproteins developed by random mutagenesis were tested by rapid colonyscreen on the substrates shown in FIGS. 18A and 18B. A rapid proteinextraction was then done, and a test of activity on alternativestructures, (e.g., as shown in FIGS. 18C-D) was performed using theprotein extract. Either the initial screen, or further screening andcharacterization of enzymes for improved activity may be performed usingother cleavage complexes, such as those diagrammed in FIGS. 21-24. It isnot intended that the scope of the invention be limited by theparticular sequences used to form such test cleavage structures. Oneskilled in the art would understand how to design and create comparablenucleic acids to form analogous structures for rapid screening.

[0704] This order of testing may be chosen to reduce the number of testsoverall, to save time and reagents. The order of testing for enzymefunction is not intended to be a limitation on the present invention.Those mutants that showed reasonable cycling rates with the RNA or DNAtargets may then be cultured overnight, and a rapid protein extractiondone. Alternatively, any subset or all of the cleavage tests may be doneat the same time.

[0705] For convenience, each type of rapid screen may be done on aseparate microtiter plate. For example, one plate may be set up to testRNA INVADER activity, one plate set up to test for DNA INVADER activity.As many as 90 different colonies may be screened on one plate. Thecolonies screened can be from a variety of sources, such as clones ofunaltered (native) 5′ nucleases, from one mutagenesis reaction (e.g.,many colonies from a single plate) or from a variety of reactions(colonies selected from multiple plates).

[0706] Ideally, positive and negative controls should be run on the sameplate as the mutants, using the same preparation of reagents. Oneexample of a good positive control is a colony containing the unmodifiedenzyme, or a previously modified enzyme whose activity is to be comparedto new mutants. For example, if a mutagenesis reaction is performed onthe Taq DN RX HT construct (described below), the unmodified Taq DN RXHT construct would be chosen as the standard for comparing the effectsof mutagenesis on enzymatic activity. Additional control enzymes mayalso be incorporated into the rapid screening test. For example, Tth DNRX HT (described below; unless otherwise specified, the TaqPol andTthPol enzymes of the following discussion refer to the DN RX HTderivative) may also be included as a standard for enzymatic activityalong with the Taq DN RX HT. This would allow a comparison of anyaltered enzymes to two known enzymes having different activities. Anegative control should also be run to determine the background reactionlevels (i.e., cleavage or probe degradation due to sources other thanthe nucleases being compared). A good negative control colony would beone containing only the vector used in the cloning and mutagenesis, forexample, colonies containing only the pTrc99A vector.

[0707] Two factors that may influence the number of colonies chosen froma specific mutagenesis reaction for the initial rapid screen are 1)total number of colonies obtained from the mutagenesis reaction, and 2)whether the mutagenesis reaction was site-specific or randomlydistributed across a whole gene or a region of a gene. For example, ifonly 5-10 colonies are present on the plate, all colonies can easily betested. If hundreds of colonies are present, a subset of these may beanalyzed. Generally 10-20 colonies are tested from a site-specificmutagenesis reaction, while 80 to 100 or more colonies are routinelytested from a single random mutagenesis reaction.

[0708] Where indicated, the altered 5′ nucleases described in theseexperimental examples were tested as detailed below.

[0709] A. Rapid Screen: INVADER Activity on RNA Target (FIG. 18A)

[0710] A 2×substrate mix was prepared, comprising 20 mM MOPS, pH 7.5, 10mM MgSO₄, 200 mM KCl, 2 μM FRET-probe oligo SEQ ID NO:223(5′-Fl-CGCT-cy3-TCTCGCTCGC-3′), 1 μM INVADER oligo SEQ ID NO:224(5′-ACGGAACGAGCGTCTTTG-3′), and 4 nM RNA target SEQ ID NO:225 (5′-GCGAGC GAGA CAG CGA AAG ACG CUC GUU CCG U-3′). Five μl of the 2×substratemix were dispensed into each sample well of a 96 well microtiter plate(Low Profile MULTIPLATE 96, M.J. Research, Inc.).

[0711] Cell suspensions were prepared by picking single colonies(mutants, positive control, and negative control colonies) andsuspending each one in 20 μl of water. This can be done conveniently ina 96 well microtiter plate format, using one well per colony.

[0712] Five μl of the cell suspension was added to the appropriate testwell such that the final reaction conditions were 10 mM MOPS, pH 7.5, 5mM MgSO₄, 100 mM KCl, 1 μM FRET-probe oligo, 0.5 μM INVADER oligo, and 2nM RNA target. The wells were covered with 10 μl of Clear CHILLOUT 14(M.J. Research, Inc.) liquid wax, and the samples were heated at 85° C.for 3 minutes, then incubated at 59° C. for 1 hour. After theincubation, the plates were read on a Cytofluor flourescense platereader using the following parameters: excitation 485/20, emission530/30.

[0713] B. Rapid Screen: INVADER Activity on DNA Target (FIG. 18B)

[0714] A 2×substrate mix was prepared, comprising 20 mM MOPS, pH 7.5, 10mM MgSO₄, 200 mM KCl, 2 μM FRET-probe oligo SEQ ID NO:223(5′-Fl-CGCT-Cy3-TCTCGCTCGC-3′), 1 μM INVADER oligo SEQ ID NO:224(5′-ACGGAACGAGCGTCTTTG-3′), 1 nM DNA target SEQ ID NO:226 (5′-GCG AGCGAGA CAG CGA AAG ACG CTC GTT CCG T-3′). Five μl of the 2×substrate mixwas dispensed into each sample well of a 96 well microtiter plate (MJLow Profile).

[0715] Cell suspensions were prepared by picking single colonies(mutants, positive control and negative control colonies) and suspendingthem in 20 μl of water, generally in a 96 well microtiter plate format.

[0716] 5 μl of the cell suspension were added to the appropriate testwell such that the final reaction conditions were 10 mM MOPS, pH 7.5, 5mM MgSO₄, 100 mM KCl, 1 μM FRET-probe oligo, 0.5 μM INVADER oligo, and0.5 nM DNA target. Wells were covered with 10 μl of Clear CHILLOUT 14(M.J. Research, Inc.) liquid wax, and the reactions were heated at 85°C. for 3 minutes, then incubated at 59° C. for 1 hour. After the hourincubation, the plate were read on a Cytofluor flourescan plate readerusing the following parameters: excitation 485/20, emission 530/30, gain40, reads per well 10.

[0717] C. Rapid Protein Extraction (Crude Cell Lysate)

[0718] Those mutants that gave a positive or an unexpected result ineither the RNA or DNA INVADER assay were further analyzed, specificallyfor background activity on the X-structure or the hairpin substrate(FIG. 18C and D, respectively). A rapid colony screen format can beemployed, as described above. By simply changing the substrate, testsfor background or aberrant enzymatic activity can be done. Anotherapproach would be to do a rapid protein extraction from a smallovernight culture of positive clones, and then test this crude celllysate for additional protein function. One possible rapid proteinextraction procedure is detailed below. Two to five ml of LB (containingthe appropriate antibiotic for plasmid selection; See e.g., Maniatis,books 1, 2 and 3) were inoculated with the remaining volume of the 20 μlwater-cell suspension and incubated at 37° C. overnight. About 1.4 ml ofthe culture were transferred to a 1.5 ml microcentrifuge tube, andmicrocentrifuged at top speed (e.g., 14,000 rpm in an Eppendorf 5417table top microcentrifuge), at room temperature for 3-5 minutes topellet the cells. The supernatant was removed, and the cell pellet wassuspended in 100 μl of TES buffer pH 7.5 (Sigma). Lysozyme (Promega) wasadded to a final concentration of 0.5 μg/l and samples were incubated atroom temperature for 30 minutes. Samples were then heated at 70° C. for10 minutes to inactivate the lysozyme, and the cell debris was pelletedby microcentrifugation at top speed for 5 minutes. The supernatant wasremoved and this crude cell lysate was used in the following enzymaticactivity assays.

[0719] D. Rapid Screen: Background Specificity X Structure Substrate(FIG. 18C)

[0720] Reactions were performed under conditions as detailed above. Oneμl of crude cell lysate was added to 9 μl of reaction components for afinal volume of 10 μl and final concentrations of 10 mM MOPS, pH 7.5, 5μM MgSO₄, 100 mM KCl, 1 μM FRET-probe oligo (SEQ ID NO:223), 0.5 μMX-structure INVADER oligo SEQ ID NO:227(5′-ACGGAACGAGCGTCTTTCATCTGTCAATC-3′), and 0.5 nM DNA target (SEQ IDNO:226). Wells were covered with 10 μl of Clear CHILLOUT 14 (M.J.Research, Inc.) liquid wax, and the reactions were heated at 85° C. for3 minutes, then incubated at 59° C. for 1 hour. After the incubation,the plates were read on a Cytofluor fluorescence plate reader using thefollowing parameters: excitation 485/20, emission 530/30, gain 40, readsper well 10.

[0721] E. Rapid Screen: Background Specificity Hairpin Substrate (FIG.18D)

[0722] Reactions were performed under conditions as detailed above. Oneμl of crude cell lysate was added to 9 μl of reaction components for afinal volume of 10 μl and final concentrations of 10 mM MOPS, pH 7.5, 5mM MgSO₄, 100 mM KCl, 1 μM FRET-probe oligonucleotide (SEQ ID NO:223),and 0.5 nM DNA target (SEQ ID NO:226). Wells were covered with 10 μl ofClear CHILLOUT 14 (M.J. Research, Inc.) liquid wax, and the reactionswere heated at 85° C. for 3 minutes, then incubated at 59° C. for 1hour. After the hour incubation, the plate were read on a Cytofluorplate reader using the following parameters: excitation 485/20, emission530/30, gain 40, reads per well 10.

[0723] F. Activity Assays with IrT1 and IdT Targets (FIGS. 24)

[0724] The 5′ nuclease activities assays were carried out in 10 μl of areaction containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05% NonidetP-40, 10 μg/ml tRNA, 100 mM KCl and 5 mM MgSO₄. The probe concentration(SEQ ID NO:167) was 2 mM. The substrates (IrTl (SEQ ID NO: 228) or IdT(SEQ ID NO: 229) at 10 or 1 nM final concentration respectively) andapproximately 20 ng of an enzyme, prepared as in Example 3, were mixedwith the above reaction buffer and overlaid with CHILLOUT (MJ Research)liquid wax. Reactions were brought up to reaction temperature 57° C.,started by addition of MgSO₄, and incubated for 10 min. Reactions werethen stopped by the addition of 10 μl of 95% formamide containing 10 MMEDTA and 0.02% methyl violet (Sigma). Samples were heated to 90° C. for1 minute immediately before electrophoresis through a 20% denaturingacrylamide gel (19:1 cross-linked), with 7 M urea, and in a buffer of 45mM Tris-borate, pH 8.3, 1.4 mM EDTA. Unless otherwise indicated, 1 μl ofeach stopped reaction was loaded per lane. Gels were then scanned on anFMBIO-100 fluorescent gel scanner (Hitachi) using a 505 nm filter. Thefraction of cleaved product was determined from intensities of bandscorresponding to uncut and cut substrate with FMBIO Analysis software(version 6.0, Hitachi). The fraction of cleavage product did not exceed20% to ensure that measurements approximated initial cleavage rates. Theturnover rate was defined as the number of cleaved signal probesgenerated per target molecule per minute under these reaction conditions(1/min).

[0725] G. Activity Assays with X Structure (X) and Hairpin (HP) Targets(FIGS. 22)

[0726] The 5′ nuclease activity assays were carried out in 10 μl of areaction containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05% NonidetP-40, 10 μg/ml tRNA, 100 mM KCl and 5 mM MgSO₄. Each oligo for formationof either the hairpin structure assembly (22A, SEQ ID NOS: 230 and 231)assembly or the X structure assembly (22B, SEQ ID NOS: 230-232) wasadded to a final concentration of 1 μm, and approximately 20 ng of testenzyme prepared as described in Example 3, were mixed with the abovereaction buffer and overlaid with CHILLOUT (MJ Research) liquid wax.Reactions were brought up to reaction temperature 60° C., started byaddition of MgSO₄, and incubated for 10 min. Reactions were then stoppedby the addition of 10 μl of 95% formamide containing 10 mM EDTA and0.02% methyl violet (Sigma). Samples were heated to 90° C. for 1 minuteimmediately before electrophoresis through a 20% denaturing acrylamidegel (19:1 cross-linked), with 7 M urea, and in a buffer of 45 mMTris-borate, pH 8.3, 1.4 mM EDTA. Unless otherwise indicated, 1 μl ofeach stopped reaction was loaded per lane. Gels were then scanned on anFMBIO-100 fluorescent gel scanner (Hitachi) using a 505 nm filter. Thefraction of cleaved product was determined from intensities of bandscorresponding to uncut and cut substrate with FMBIO Analysis software(version 6.0, Hitachi). The fraction of cleavage product did not exceed20% to ensure that measurements approximated initial cleavage rates. Theturnover rate was defined as the number of cleaved signal probesgenerated per target molecule per minute under these reaction conditions(1/min).

[0727] H. Activity Assays with Human IL-6 Target (FIG. 10)

[0728] The 5′ nuclease activities assays were carried out in 10 plreactions containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05% NonidetP-40, 10 μg/ml tRNA, 100 mM KCl and 5 mM MgSO₄. Reactions comprising theDNA IL-6 substrate contained 0.05 nM IL-6 DNA target (SEQ ID NO:163) and1 μM of each probe (SEQ ID NO:162) and INVADER (SEQ ID NO:161)oligonucleotides, and were carried out at 60° C. for 30 min. Reactionscomprising the IL-6 RNA target (SEQ ID NO:160) were performed under thesame conditions, except that the IL-6 RNA target concentration was 1 nMand the reactions were performed at 57° C. for 60 min. Each reactioncontained approximately 20 ng of test enzyme, prepared as described inExample 3.

[0729] I. Activity Assays with Synthetic r25mer Target (FIG. 23)

[0730] Reactions comprising the synthetic r25mer target (SEQ ID NO: 233)were carried 30 out under the same reaction conditions (10 mM MOPS, pH7.5, 0.05% Tween 20, 0.05% Nonidet P-40, 10 μg/ml tRNA, 100 mM KCl and 5mM MgSO₄) and 1 μM of each probe (SEQ ID NO: 234) and INVADER (SEQ IDNO: 235) oligonucleotides, except that the r25mer target concentrationwas 5 nM and the reactions were performed at 58° C. for 60 min.Approximately 20 ng of each test enzyme was added to the reactions.Enzymes were prepared as described in Example 3.

[0731] Any of the tests described above can be modified to derive theoptimal conditions for enzymatic activity. For example, enzymetitrations can be done to determine the optimal enzyme concentration formaximum cleavage activity, and lowest background signal. By way ofexample, but not by way of limitation, many of the mutant enzymes weretested at 10, 20 and 40 ng amounts. Similarly, a temperature titrationcan also be incorporated into the tests. Since modifying the structureof a protein can alter its temperature requirements, a range oftemperatures can be tested to identify the condition best suited for themutant in question. Additionally, different buffer conditions may betested. Since modifying the structure of a protein can alter itsrequirements for salts (e.g., KCl), a range of salt (or other buffercomponent) concentrations can be tested to identify the conditions bestsuited for the mutant in question. In some embodiments, enzymes aretested or used in conditions that do not comprise salt added as a buffercomponent (although some salt may be present from sample preparation orenzyme preparation).

[0732] Examples of the results from such screens (using approximately 20ng of the mutant enzyme) are shown in Tables 3-8, and FIGS. 12, 14, 15,19, and 25.

Example 2 Cloning and Expression of 5′ Nucleases of DNA Polymerases andMutant Polymerases

[0733] A. DNA Polymerases of Thermus aquaticus and Thermus thermophilus

[0734] 1. Cloning of TaqPol and TthPol

[0735] Type A DNA polymerases from eubacteria of the genus Thermus shareextensive protein sequence identity (90% in the polymerization domain,using the Lipman-Pearson method in the DNA analysis software fromDNAStar, Wis.) and behave similarly in both polymerization and nucleaseassays. Therefore, the genes for the DNA polymerase of Thermus aquaticus(TaqPol), Thermus thermophilus (TthPol) and Thermus scotoductus wereused as representatives of this class. Polymerase genes from othereubacterial organisms, including, but not limited to, Escherichia coli,Streptococcus pneumoniae, Mycobacterium smegmatis, Thermus thermophilus,Thermus sp., Thermotoga maritima, Thermosipho africanus, and Bacillusstearothermophilus are equally suitable.

[0736] a. Initial TaqPol Isolation: Mutant TaqA/G

[0737] The Taq DNA polymerase gene was amplified by polymerase chainreaction from genomic DNA from Thermus aquaticus, strain YT-1 (Lawyer etal, supra), using as primers the oligonucleotides described in SEQ IDNOS:236 and 237. The resulting fragment of DNA has a recognitionsequence for the restriction endonuclease EcoRI at the 5′ end of thecoding sequence and a BglII sequence at the 3′ end of the coding strand.Cleavage with BglII leaves a 5′ overhang or “sticky end” that iscompatible with the end generated by BamHI. The PCR-amplified DNA wasdigested with EcoRI and BamHI. The 2512 bp fragment containing thecoding region for the polymerase gene was gel purified and then ligatedinto a plasmid that contains an inducible promoter.

[0738] In one embodiment of the invention, the pTTQ18 vector, whichcontains the hybrid trp-lac (tac) promoter, was used (M. J. R. Stark,Gene 5:255 [1987]). The tac promoter is under the control of the E. colilac repressor protein. Repression allows the synthesis of the geneproduct to be suppressed until the desired level of bacterial growth hasbeen achieved, at which point repression is removed by addition of aspecific inducer, isopropyl-b-D-thiogalactopyranoside (IPTG). Such asystem allows the controlled expression of foreign proteins that mayslow or prevent growth of transformants.

[0739] Particularly strong bacterial promoters, such as the syntheticPtac, may not be adequately suppressed when present on a multiple copyplasmid. If a highly toxic protein is placed under control of such apromoter, the small amount of expression leaking through, even in theabsence of an inducer, can be harmful to the bacteria. In anotherembodiment of the invention, another option for repressing synthesis ofa cloned gene product is contemplated. A non-bacterial promoter frombacteriophage T7, found in the plasmid vector series pET-3, was used toexpress the cloned mutant Taq polymerase genes (Studier and Moffatt, J.Mol. Biol., 189:113 [1986]). This promoter initiates transcription onlyby T7 RNA polymerase. In a suitable strain, such as BL21(DE3)pLYS, thegene for the phage T7 RNA polymerase is carried on the bacterial genomeunder control of the lac operator. This arrangement has the advantagethat expression of the multiple copy gene (on the plasmid) is completelydependent on the expression of T7 RNA polymerase, which is easilysuppressed because it is present in a single copy.

[0740] These are just two examples of vectors having suitable induciblepromoters. Others are well known to those skilled in the art, and it isnot intended that the improved nucleases of the present invention belimited by the choice of expression system.

[0741] For ligation into the pTTQ18 vector, the PCR product DNAcontaining the Taq polymerase coding region (termed mutTaq for reasonsdiscussed below, SEQ ID NO:238) was digested with EcoRI and BglII andthis fragment was ligated under standard “sticky end” conditions(Sambrook et al Molecular Cloning, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, pp. 1.63-1.69 [1989]) into the EcoRI and BamHI sitesof the plasmid vector pTTQ18. Expression of this construct yields atranslational fusion product in which the first two residues of thenative protein (Met-Arg) are replaced by three from the vector(Met-Asn-Ser), but the remainder of the PCR product's protein sequenceis not changed (SEQ ID NO:239). The construct was transformed into theJM109 strain of E. coli, and the transformants were plated underincompletely repressing conditions that do not permit growth of bacteriaexpressing the native protein. These plating conditions allow theisolation of genes containing pre-existing mutations, such as those thatresult from the infidelity of Taq polymerase during the amplificationprocess.

[0742] Using this amplification/selection protocol, a clone was isolatedcontaining a mutated Taq polymerase gene (mutTaq). The mutant was firstdetected by its phenotype, in which temperature-stable 5′ nucleaseactivity in a crude cell extract was normal, but polymerization activitywas almost absent (approximately less than 1% of wild type Taqpolymerase activity). Polymerase activity was determined by primerextension reactions. The reactions were carried out in 10 μl of buffercontaining 10 mM MOPS, pH 7.5, 5 mM MgSO₄, 100 mM KCl. In each reaction,40 ng of enzyme were used to extend 10 μM (dT)₂₅₋₃₀ primer in thepreesnce of either 10 μM poly (A)₂₈₆ or 1 μM poly (dA)₂₇₃ template, 45μM dTTP and 5 μM Fl-dUTP at 60° C. for 30 minutes. Reactions werestopped with 10 μl of stop solution (95% formamide, 10 mM EDTA, 0.02%methyl violet dye). Samples (3 μl) were fractionated on a 15% denaturingacrylamide gel (19:1 crossed-linked) and the fraction of incorporatedFl-dUTP was quantitated using an FMBIO-100 fluorescence gel scanner(Hitachi) equipped with a 505 nm emission filter.

[0743] DNA sequence analysis of the recombinant gene showed that it hadchanges in the polymerase domain resulting in two amino acidsubstitutions: an A to G change at nucleotide position 1394, whichcauses a Glu to Gly change at amino acid position 465 (numberedaccording to the natural nucleic and amino acid sequences, SEQ IDNOS:153 and 157), and another A to G change at nucleotide position 2260,which causes a Gln to Arg change at amino acid position 754. Because theGln to Gly mutation is at a nonconserved position and because the Glu toArg mutation alters an amino acid that is conserved in virtually all ofthe known Type A polymerases, the latter mutation is most likely the oneresponsible for curtailing the synthesis activity of this protein. Thenucleotide sequence for the construct is given in SEQ ID NO:39. Theenzyme encoded by this sequence is referred to as Taq A/G.

[0744] b. Initial TthPol Isolation

[0745] The DNA polymerase enzyme from the bacterial species Thermusthermophilus (Tth) was produced by cloning the gene for this proteininto an expression vector and overproducing it in E. coli cells. GenomicDNA was prepared from 1 vial of dried Thermus thermophilus strain HB-8from ATCC (ATCC #27634). The DNA polymerase gene was amplified by PCRusing the following primers: 5′-CACGAATTCCGAGGCGATGCTTCCGCTC-3′ (SEQ IDNO:240) and 5′-TCGACGTCGACTAACCCTTGGCGGAAAGCC-3′ (SEQ ID NO:241). Theresulting PCR product was digested with EcoRI and SalI restrictionendonucleases and inserted into EcoRI/Sal I digested plasmid vectorpTrc99G (described in Example 2C1) to create the plasmid pTrcTth-1. ThisTth polymerase construct is missing a single nucleotide that wasinadvertently omitted from the 5′ oligonucleotide, resulting in thepolymerase gene being out of frame. This mistake was corrected by sitespecific mutagenesis of pTrcTth-1 as described in Examples 4 and 5 usingthe following oligonucleotide: 5′-GCATCGCCTCGGAATTCATGGTC-3′ (SEQ IDNO:242), to create the plasmid pTrcTth-2. The protein and the nucleicacid sequence encoding the protein are referred to as TthPol, and arelisted as SEQ ID NOS:243 and 244 respectively.

[0746] C. Large Scale Preparation of Recombinant Proteins

[0747] The recombinant proteins were purified by the following techniquewhich is derived from a Taq DNA polymerase preparation protocol (Engelkeet al., Anal. Biochem., 191:396 [1990]) as follows. E. coli cells(strain JM109) containing either pTrc99A TaqPol, pTrc99GTthPol wereinoculated into 3 ml of LB containing 100 mg/ml ampicillin and grown for16 hrs at 37° C. The entire overnight culture was inoculated into 200 mlor 350 ml of LB containing 100 mg/ml ampicillin and grown at 37° C. withvigorous shaking to an A₆₀₀ of 0.8. IPTG (1 M stock solution) was addedto a final concentration of 1 mM and growth was continued for 16 hrs at37° C.

[0748] The induced cells were pelleted and the cell pellet was weighed.An equal volume of 2×DG buffer (100 mM Tris-HCl, pH 7.6, 0.1 mM EDTA)was added and the pellet was suspended by agitation. Fifty mg/mllysozyme (Sigma) were added to 1 mg/ml final concentration and the cellsincubated at room temperature for 15 min. Deoxycholic acid (10%solution) was added dropwise to a final concentration of 0.2% whilevortexing. One volume of H₂O and 1 volume of 2×DG buffer were added, andthe resulting mixture was sonicated for 2 minutes on ice to reduce theviscosity of the mixture. After sonication, 3 M (NH₄)₂SO₄ was added to afinal concentration of 0.2 M, and the lysate was centrifuged at 14000×gfor 20 min at 4° C. The supernatant was removed and incubated at 70° C.for 60 min at which time 10% polyethylimine (PEI) was added to 0.25%.After incubation on ice for 30 min., the mixture was centrifuged at14,000×g for 20 min at 4° C. At this point, the supernatant was removedand the protein precipitated by the addition of (NH₄)₂SO₄ as follows.

[0749] Two volumes of 3 M (NH₄)₂SO₄ were added to precipitate theprotein. The mixture was incubated overnight at room temperature for 16hrs centrifuged at 14,000×g for 20 min at 4° C. The protein pellet wassuspended in 0.5 ml of Q buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA,0.1% Tween 20). For the Mja FEN-1 preparation, solid (NH₄)₂SO₄ was addedto a final concentration of 3 M (˜75% saturated), the mixture wasincubated on ice for 30 min, and the protein was spun down and suspendedas described above.

[0750] The suspended protein preparations were quantitated bydetermination of the A₂₇₉ dialyzed and stored in 50% glycerol, 20 mMTris HCl, pH 8.0, 50 mM KCl, 0.5% Tween 20, 0.5% Nonidet P-40, with 100μg/ml BSA.

[0751] B. DNA polymerases of Thermus filiformis and Thermus scotoductus

[0752] 1. Cloning of Thermus filiformis and Thermus scotoductus

[0753] One vial of lyophilized Thermus filiformis (Tfi) obtained fromDSMZ (Deutsche Sammlung von Mikroorganismen und Zellculturen,Braunschweig, Germany, strain #4687) was rehydrated in 1 ml ofCastenholz medium (DSMZ medium 86) and inoculated into 500 ml ofCastenholz medium preheated to 50° C. The culture was incubated at 70°C. with vigorous shaking for 48 hours. After growth, the cells wereharvested by centrifugation at 8000×g for 10 minutes, the cell pelletwas suspended in 10 ml of TE (10 mM Tris HCL, pH 8.0, 1 mM EDTA), andthe cells were frozen at −20° C. in 1 ml aliquots. A 1 ml aliquot wasthawed, lysozyme was added to 1 mg/ml, and the cells were incubated at23° C. for 30 minutes. A solution of 20% SDS (sodium dodecyl sulfate)was added to a final concentration of 0.5% followed by extraction withbuffered phenol. The aqueous phase was further extracted with 1:1phenol:chloroform, and extracted a final time with chloroform. One-tenthvolume of 3 M sodium acetate, pH 5.0 and 2.5 volumes of ethanol wereadded to the aqueous phase and mixed. The DNA was pelleted bycentrifugation at 20,000×g for 5 minutes. The DNA pellet was washed with70% ethanol, air dried and resuspended in 200 μl of TE and used directlyfor amplification. Thermus scotoductus (Tsc, ATCC # 51532) was grown andgenomic DNA was prepared as described above for Thermus filiformis.

[0754] The DNA polymerase I gene from Tfi (GenBank accession #AF030320)could not be amplified as a single fragment. Therefore, it was cloned in2 separate fragments into the expression vector pTrc99a. The 2 fragmentsoverlap and share a Not I site which was created by introducing a silentmutation at position 1308 of the Tfi DNA polymerase open reading frame(ORF) in the PCR oligonucleotides. The 3′ half of the gene was amplifiedusing the Advantage cDNA PCR kit (Clonetech) with the followingoligonucleotides; 5′-ATAGCCATGGTGGAGCGGCCGCTCTCCCGG (SEQ ID NO:245) and5′-AAGCGTCGACTCAATCCTGCTTCGCCTCCAGCC (SEQ ID NO:246). The PCR productfrom this reaction was approximately 1200 base pairs in length. It wascut with the restriction enzymes Not I and Sal I, and the resulting DNAwas ligated into pTrc99a cut with NotI and SalI to create pTrc99a-Tfi3′.The 5′ half of the gene was amplified as described above using thefollowing two primers; 5′AATCGAATTCACCCCACTTTTTGACCTGGAGG (SEQ IDNO:247) and 5′-CCGGGAGAGCGGCCGCTCCAC (SEQ ID NO:248). The resulting 1300base pair fragment was cut with restriction enzymes Eco RI and Not I andligated into pTrc99a-Tfi3′ cut with NotI and EcoRI to producepTrc99a-TfiPol, SEQ ID NO:249 (the corresponding amino acid sequence islisted in SEQ ID NO:250).

[0755] The DNA polymerase I gene from Thermus scotoductus was amplifiedusing the Advantage cDNA PCR kit (Clonetech) using the following twoprimers; 5′-ACTGGAATTCCTGCCCCTCTTTGAGCCCAAG (SEQ ID NO:251) and5′-AACAGTCGACCTAGGCCTTGGCGGAAAGCC (SEQ ID NO:252). The PCR product wascut with restriction enzymes Eco RI and Sal I and ligated into Eco RI,Sal I cut pTrc99a to create pTrc99a-TscPol SEQ ID NO:253 (thecorresponding amino acid sequence is listed in SEQ ID NO:254).

[0756] 2. Expression and Purification of Thermus filiformis and Thermusscotoductus

[0757] Plasmids were transformed into protease deficient E. coli strainBL21 (Novagen) or strain JM109 (Promega Corp., Madison, Wis.) forprotein expression. Flasks containing 200 ml of LB containing 100 μg/mlampicillin were inoculated with either a single colony from an LB plateor from a frozen stock of the appropriate strain. After several hours ofgrowth at 37° C. with vigorous shaking, cultures was induced by theaddition of 200 μl of 1 M isothiopropyl-galatoside (IPTG). Growth at 37°C. was continued for 16 hours prior to harvest. Cells were pelleted bycentrifugation at 8000×g for 15 minutes followed by suspension of thecell pellet in 5 ml of TEN (10 mM Tris HCl, pH 8.0, 1 mM EDTA, 100 mMNaCl). 100 μl of 50 mg/ml lysozyme were added and the cells incubated atroom temperature for 15 minutes. Deoxycholic acid (10%) was added to afinal concentration of 0.2%. After thorough mixing, the cell lysateswere sonicated for 2 minutes on ice to reduce the viscosity of themixture. Cellular debris was pelleted by centrifugation at 4° C. for 15minutes at 20,000×g. The supernatant was removed and incubated at 70° C.for 30 min after which 10% polyethylimine (PEI) was added to 0.25%.After incubation on ice for 30 minutes, the mixture was centrifuged at20,000×g for 20 min at 4° C. At this point, the supernatant containingthe enzyme was removed, and the protein was precipitated by the additionof 1.2 g of ammonium sulfate and incubation at 4° C. for 1 hour. Theprotein was pelleted by centrifugation at 4° C. for 10 minutes at20,000×g. The pellet was resuspended in 4 ml of HPLC Buffer A (50 mMTris HCl, pH 8.0, 1 mM EDTA). The protein was further purified byaffinity chromatography using an Econo-Pac heparin cartridge (Bio-Rad)and a Dionex DX 500 HPLC instrument. Briefly, the cartridge wasequilibrated with HPLC Buffer A, and the enzyme extract was loaded onthe column and eluted with a linear gradient of NaCl (0-2 M) in the samebuffer. Pure protein elutes between 0.5 and 1 M NaCl. The enzyme peakwas collected and dialyzed in 50% glycerol, 20 mM Tris HCl, pH 8, 50 mMKCl, 0.5% Tween 20, 0.5% Nonidet P40, 100 mg/ml BSA.

[0758] C. Generation of Polymerase Mutants with Reduced PolymeraseActivity but Unaltered 5′ Nuclease Activity

[0759] All mutants generated in section C were expressed and purified asdescribed in Example 2A1C.

[0760] 1. Modified TaqPol Genes: TaqDN

[0761] A polymerization deficient mutant of Taq DNA polymerase calledTaqDN was constructed. TaqDN nuclease contains an asparagine residue inplace of the wild-type aspartic acid residue at position 785 (D785N).

[0762] DNA encoding the TaqDN nuclease was constructed from the geneencoding the Taq A/G in two rounds of site-directed mutagenesis. First,the G at position 1397 and the G at position 2264 of the Taq A/G gene(SEQ ID NO:238) were changed to A at each position to recreate awild-type TaqPol gene. In a second round of mutagenesis, the wild typeTaqPol gene was converted to the Taq DN gene by changing the G atposition 2356 to A. These manipulations were performed as follows.

[0763] DNA encoding the Taq A/G nuclease was recloned from pTTQ18plasmid into the pTrc99A plasmid (Pharmacia) in a two-step procedure.First, the pTrc99A vector was modified by removing the G at position 270of the pTrc99A map, creating the pTrc99G cloning vector. To this end,pTrc99A plasmid DNA was cut with NcoI and the recessive 3′ ends werefilled-in using the Klenow fragment of E. coli polymerase I in thepresence of all four dNTPs at 37° C. for 15 min. After inactivation ofthe Klenow fragment by incubation at 65° C. for 10 min, the plasmid DNAwas cut with EcoRI and the ends were again filled-in using the Klenowfragment in the presence of all four dNTPs at 37° C. for 15 min. TheKlenow fragment was then inactivated by incubation at 65° C. for 10 min.The plasmid DNA was ethanol precipitated, recircularized by ligation,and used to transform E.coli JM109 cells (Promega). Plasmid DNA wasisolated from single colonies, and deletion of the G at position 270 ofthe pTrc99A map was confirmed by DNA sequencing.

[0764] In a second step, DNA encoding the Taq A/G nuclease was removedfrom the pTTQ18 plasmid using EcoRI and SalI and the DNA fragmentcarrying the Taq A/G nuclease gene was separated on a 1% agarose gel andisolated with Geneclean II Kit (Bio 101, Vista, Calif.). The purifiedfragment was ligated into the pTrc99G vector that had been cut withEcoRI and SalI. The ligation mixture was used to transform competent E.coli JM109 cells (Promega). Plasmid DNA was isolated from singlecolonies and insertion of the Taq A/G nuclease gene was confirmed byrestriction analysis using EcoRI and SalI.

[0765] Plasmid DNA pTrcAG carrying the Taq A/G nuclease gene cloned intothe pTrc99A vector was purified from 200 ml of JM109 overnight cultureusing QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, Calif.) according tomanufacturer's protocol. pTrcAG plasmid DNA was mutagenized using twomutagenic primers, E465 (SEQ ID NO:255) (Integrated DNA Technologies,Iowa) and R754Q (SEQ ID NO:256) (Integrated DNA Technologies), and theselection primer Trans Oligonucleotide AlwNI/SpeI (Clontech, Palo Alto,Calif., catalog #6488-1) according to TRANSFORMER Site-DirectedMutagenesis Kit protocol (Clontech, Palo Alto, Calif.) to produce arestored wild-type TaqPol gene (pTrcWT).

[0766] pTrcWT plasmid DNA carrying the wild-type TaqPol gene cloned intothe pTrc99A vector was purified from 200 ml of JM109 overnight cultureusing QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, Calif.) according tomanufacturer's protocol. pTrcWT was then mutagenized using the mutagenicprimer D785N (SEQ ID NO:257) (Integrated DNA Technologies) and theselection primer Switch Oligonucleotide SpeI/AlwNI (Clontech, Palo Alto,Calif., catalog #6373-1) according to TRANSFORMER Site-DirectedMutagenesis Kit protocol (Clontech, Palo Alto, Calif.) to create aplasmid containing DNA encoding the Taq DN nuclease. The DNA sequenceencoding the Taq DN nuclease is provided in SEQ ID NO:258; the aminoacid sequence of Taq DN nuclease is provided in SEQ ID NO:259.

[0767] 2. Modified TthPol Gene: Tth DN

[0768] The Tth DN construct was created by mutating the TthPol describedabove. The sequence encoding an aspartic acid at position 787 waschanged by site-specific mutagenesis as described above to a sequenceencoding asparagine. Mutagenesis of pTrcTth-2 with the followingoligonucleotide: 5′-CAGGAGGAGCTCGTTGTGGACCTGGA-3′ (SEQ ID NO:260) wasperformed to create the plasmid pTrcTthDN. The mutant protein andprotein coding nucleic acid sequence is termed TthDN SEQ ID NOS:261 and262 respectively.

[0769] 3. Taq DN HT and Tth DN HT

[0770] Six amino acid histidine tags (his-tags) were added onto thecarboxy termini of Taq DN and Tth DN. The site-directed mutagenesis wasperformed using the TRANSFORMER Site Directed Mutagenesis Kit (Clontech)according to the manufacturer's instructions. The mutagenicoligonucleotides used on the plasmids pTaq DN and pTth DN were sequence117-067-03, 5′-TCTAGAGGATCTATCAGTGGTGGTGGTGGTGGTGCTCCTTGGCGGAGAGC-3′(SEQ ID NO:263) and5′-TGCCTGCAGGTCGACGCTAGCTAGTGGTGGTGGTGGTGGTGACCCTTGGCG GAAAGCC-3′ (SEQID NO:264), sequence 136-037-05. The selection primer Trans OligoAlwNI/SpeI (Clontech, catalog # 6488-1) was used for both mutagenesisreactions. The resulting mutant genes were termed Taq DN HT (SEQ IDNO:265, nucleic acid sequence; SEQ ID NO:266, amino acid sequence) andTth DN HT (SEQ ID NO:267, nucleic acid sequence; SEQ ID NO:268, aminoacid sequence).

[0771] 4. Purification of Taq DN HT and Tth DN HT

[0772] Both Taq DN HT and Tth DN HT proteins were expressed in E. colistrain JM109 as described in Example 2B2. After ammonium sulfateprecipitation and centrifugation, the protein pellet was suspended in0.5 ml of Q buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTAm 0.1% Tween 20).The protein was further purified by affinity chromatography usingHis-Bind Resin and Buffer Kit (Novagen) according to the manufacturer'sinstructions. 1 ml of His-Bind resin was transferred into a column,washed with 3 column volumes of sterile water, charged with 5 volumes of1×Charge Buffer, and equilibrated with 3 volumes of 1×Binding Buffer.Four ml of 1×Binding Buffer was added to the protein sample and thesample solution was loaded onto the column. After washing with 3 ml of1×Binding Buffer and 3 ml of 1×Wash Buffer, the bound His-Tag proteinwas eluted with 1 ml of 1×Elute Buffer. The pure enzyme was thendialyzed in 50% glycerol, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.5% Tween20, 0.5% Nonidet P40, and 100 μg.ml BSA. Enzyme concentrations weredetermined by measuring absorption at 279 nm.

Example 3 RNA-Dependent 5′ Nuclease Activity of TthPol can be Conferredon TaqPol by Transfer of the N-Terminal Portion of the DNA PolymeraseDomain

[0773] A. Preparation and Purification of Substrate Structures HavingEither a DNA or an RNA Target Strand

[0774] The downstream (SEQ ID NO:162) and upstream probes (SEQ IDNO:161) and the IL-6 DNA (SEQ ID NO:163) (FIG. 10) target strand weresynthesized on a PerSeptive Biosystems instrument using standardphosphoramidite chemistry (Glen Research). The synthetic RNA-DNAchimeric IrT target labeled with biotin at the 5′-end (FIG. 20A) wassynthesized utilizing 2′-ACE RNA chemistry (Dharmacon Research). The2′-protecting groups were removed by acid-catalyzed hydrolysis accordingto the manufacturer's instructions. The downstream probes labeled with5′-fluorescein (Fl) or 5′-tetrachloro-fluorescein (TET) at their 5′ endswere purified by reverse phase HPLC using a Resource Q column(Amersham-Pharmacia Biotech). The 648-nucleotide IL-6 RNA target (SEQ IDNO:160) (FIG. 10) was synthesized by T7 RNA polymeraserunoff-transcription of the cloned fragment of human IL-6 cDNA(nucleotides 64-691 of the sequence published in May et al., Proc. Natl.Acad. Sci., 83:8957 [1986]) using a Megascript Kit (Ambion). Alloligonucleotides were finally purified by separation on a 20% denaturingpolyacrylamide gel followed by excision and elution of the major band.Oligonucleotide concentration was determined by measuring absorption at260 nm. The biotin labeled IrT target was incubated with a 5-fold excessof streptavidin (Promega) in a buffer containing 10 mM MOPS, pH 7.5,0.05% Tween 20, 0.05% NP-40 and 10 μg/ml tRNA at room temperature for 10min.

[0775] B. Introduction of Restriction Sites to Make Chimeras

[0776] The restriction sites used for formation of chimerical proteins,described below, were chosen for convenience. The restriction sites inthe following example have been strategically placed to surround regionsshown by crystal structure and other analysis to be functional domains(See, FIGS. 6, 7, and 19). Different sites, either naturally occurringor created via directed mutagenesis can be used to make similarconstructs with other Type A polymerase genes from related organisms. Itis desirable that the mutations all be silent with respect to proteinfunction. By studying the nucleic acid sequence and the amino acidsequence of the protein, one can introduce changes in the nucleic acidsequence that have no effect on the corresponding amino acid sequence.If the nucleic acid change required affects an amino acid, one can makethe alteration such that the new amino acid has the same or similarcharacteristics of the one replaced. If neither of these options ispossible, one can test the mutant enzyme for function to determine ifthe nucleic acid alteration caused a change in protein activity,specificity or function. It is not intended that the invention belimited by the particular restriction sites selected or introduced forthe creation of the improved enzymes of the present invention.

[0777] C. Generation of Tth DN RX HT and Taq DN RX HT

[0778] Mutagenesis was performed to introduce 3 additional, uniquerestriction sites into the polymerase domain of both the Taq DN HT andTth DN HT enzymes. Site-specific mutagenesis was performed using theTransformer Site-Directed Mutagenesis Kit from (Clonetech) according tomanufacturer's instructions. One of two different selection primers,Trans Oligo AlwNI/SpeI or Switch Oligo SpeI/AlwNI (Clontech, Palo AltoCalif. catalog #6488-1 or catalog #6373-1) was used for all mutagenesisreactions described. The selection oligo used in a given reaction isdependent on the selection restriction site present in the vector. Allmutagenic primers were synthesized by standard synthetic chemistry.Resultant colonies were expressed in E. coli strain JM 109.

[0779] The Not I sites (amino acid position 328) were created using themutagenic primers 5′-gccgccaggggcggccgcgtccaccgggcc (SEQ ID NO:269) and5′-gcctgcaggggcggccgcgtgcaccggggca (SEQ ID NO:270) corresponding to thesense strands of the Taq DN HT and the Tth DN HT genes, respectively.The BstI (amino acid position 382) and NdeI (amino acid position 443)sites were introduced into both genes using sense strand mutagenicprimes 5′-ctcctggacccttcgaacaccacccc (SEQ ID NO:271) and5′-gtcctggcccatatggaggccac (SEQ ID NO:272). The mutant plasmids wereover-expressed and purified using Qiagen QiaPrep Spin Mini Prep Kit(cat. # 27106). The vectors were tested for the presence of therestriction sites by DNA sequencing and restriction mapping. Theseconstructs are termed Tth DN RX HT (DNA sequence SEQ ID NO:273; aminoacid sequence SEQ ID NO:274) and Taq DN RX HT (DNA sequence SEQ IDNO:275; amino acid sequence SEQ ID NO:276).

[0780] D. Chimeras

[0781] The chimeric constructs shown in FIG. 19 were created byexchanging homologous DNA fragments defined by the restrictionendonuclease sites EcoRI (E) and BamHI (B), common for both genes, thecloning vector site SalI (S) and the new sites, NotI (N), BstBI (Bs),NdeI (D) created at the homologous positions of both genes by sitedirected mutagenesis. In generating these chimeric enzymes, twodifferent pieces of DNA are ligated together to yield the finalconstruct. The larger piece of DNA that contains the plasmid vector aswell as part of the Taq or Tth (or parts of both) sequence will betermed the “vector.” The smaller piece of DNA that contains sequences ofeither the Taq or Tth (or parts of both) polymerase will be termed the“insert.”

[0782] All restriction enzymes were from New England Biolabs or Promegaand used in reactions with the accompanying buffer, according to themanufacturer's instructions. Reactions were done in 20 μl volume withabout 500 ng of DNA per reaction, at the optimal temperature for thespecified enzyme. More than one enzyme was used in a single reaction(double digest) if the enzymes were compatible with respect to reactionbuffer conditions and reaction temperature. If the enzymes in questionwere not compatible with respect to buffer conditions, the enzymerequiring the lowest salt condition was used first. After the completionof that reaction, buffer conditions were changed to be optimal or bettersuited to the second enzyme, and the second reaction was performed.These are common restriction enzyme digest strategies, well known tothose in the art of basic molecular biology (Maniatis, supra).

[0783] The digested restriction fragments were gel isolated for optimalligation efficiency. Two μl of 10×loading dye (50% glycerol, 1×TAE, 0.5%bromophenol blue) were added to the 20 μl reaction. The entire volumewas loaded and run on a 1%, 1×TAE agarose gel containing 1 μl of a 1%ethidium bromide solution per 100 ml of agarose gel solution. Thedigested fragments were visualized under UV light, and the appropriatefragments (as determined by size) were excised from the gel. Thesefragments were then purified using the Qiagen Gel Extractio Kit, (cat #28706) according to the manufacturer's instructions.

[0784] Ligations were performed in a 10 μl volume, using 400 units perreaction of T4 DNA Ligase enzyme from New England Biolabs (catalog#202L), with the accompanying reaction buffer. Ligation reactions weredone at room temperature for 1 hour, with 1 μl of each of theQiagen-purified fragments (approximately 20-50 ng of each DNA, dependingon recovery from the gel isolation). Ligation products were thentransformed into E. coli strain JM 109 and plated onto an appropriategrowth and selection medium, such as LB with 100 μg/ml of ampicillin toselect for transformants.

[0785] For each ligation reaction, six transformants were tested todetermine if the desired construct was present. Plasmid DNA was purifiedand isolated using the QiaPrep Spin Mini Prep Kit, according tomanufacturer's instructions. The constructs were verified by DNAsequencing and by restriction mapping.

[0786] Expression and purification of the chimeric enzymes was done asfollows. Plasmids were transformed into E. coli strain JM109 (Promega).Log phase cultures (200 ml) of JM109 were induced with 0.5 mM IPTG(Promega) and grown for an additional 16 hours prior to harvest. Crudeextracts containing soluble proteins were prepared by lysis of pelletedcells in 5 ml of 10 mM Tris-HCl, pH 8.3, 1 mM EDTA, 0.5 mg/ml lysozymeduring incubation at room temperature for 15 minutes. The lysate wasmixed with 5 ml of 10 mM Tris-HCl pH 7.8, 50 mM KCl, 1 mM EDTA, 0.5%Tween 20, 0.5% Nonidet P-40, heated at 72° C. for 30 minutes, and celldebris was removed by centrifugation at 12,000×g for 5 minutes. Finalpurification of the protein was done by affinity chromatograpy using anEcono-Pac heparin cartridge (Bio-Rad) and Dionex DX 500 HPLC instrument.Briefly, the cartridge was equilibrated with 50 mM Tris-HCl pH 8, 1 mMEDTA, and an enzyme extract dialyzed against the same buffer was loadedon the column and eluted with a linear gradient of NaCl (0-2 M) in thesame buffer. The HPLC-purified protein was dialyzed and stored in 50%(vol/vol) glycerol, 20 mM Tris-HCl pH 8.0, 50 mM KCl, 0.5% Tween 20,0.5% Nonidet P-40, and 100 μg/m BSA. The enzymes were purified tohomogeneity according to SDS-PAGE, and the enzyme concentrations weredetermined by measuring absorption at 279 nm.

[0787] 1. Construction of TaqTth(N) and TthTaq(N)

[0788] The first exchange that was performed involved the polymerasedomains of the two enzymes. Separation of the nuclease domain (theN-terminal end of the protein) from the polymerase domain (theC-terminal portion of the protein) was accomplished by cutting bothgenes with the restriction endonucleases EcoRI and NotI. Theapproximately 900 base pair fragment from the Tth DN RX HT gene wascloned into the homologous sites of the Taq DN RX HT gene, and theapproximately 900 base pair fragment from the Taq DN RX HT gene wascloned into the homologous sites of the Tth DN RX HT gene, yielding twochimeras, TaqTth(N) (DNA sequence SEQ ID NO:69; amino acid sequence SEQID NO:2) which has the Taq DN RX HT 5′ nuclease domain and the Tth DN RXHT polymerase domain, and TthTaq(N) (DNA sequence SEQ ID NO:70; aminoacid sequence SEQ ID NO:3) which is made up of the Tth DN RX HT 5′nuclease domain and the Taq DN RX HT polymerase domain.

[0789] 2. Construction of TaqTth(N-B)

[0790] The Taq DN RX HT construct was cut with the enzymes NdeI andBamHI and the larger, vector fragment was gel isolated as detailedabove. The Tth DN RX HT construct was also cut with NdeI and BamHI andthe smaller (approximately 795 base pairs) Tth fragment was gel isolatedand purified. The Tth NdeI-BamHI insert was ligated into the TaqNdeI-BamHI vector as detailed above to generate the TaqTth(N-B) (DNAsequence SEQ ID NO:71; amino acid sequence SEQ ID NO:4).

[0791] 3. Construction of TaqTth(B-S)

[0792] The Taq DN RX HT construct was cut with the enzymes BamHI andSalI and the larger vector fragment was gel isolated as detailed above.The Tth DN RX HT construct was also cut with BamHI and SalI and thesmaller (approximately 741 base pairs) Tth fragment was gel isolated andpurified. The Tth BamHI-SalI insert was ligated into the Taq BamHI-SalIvector as detailed above to generate the TaqTth(B-S) (DNA sequence SEQID NO:72; amino acid sequence SEQ ID NO:5).

[0793] 4. Construction of TaqTth(N-D)

[0794] The Taq DN RX HT construct was cut with the enzymes NotI and NdeIand the larger vector fragment was isolated as detailed above. The TthDN RX HT construct was also cut with NotI and NdeI and the smaller(approximately 345 base pairs) Tth fragment was gel isolated andpurified. The Tth NotI-NdeI insert was ligated into the Taq NotI-NdeIvector as detailed above to generate the TaqTth(N-D) (DNA sequence SEQID NO:73; amino acid sequence SEQ ID NO:6).

[0795] 5. Construction of TaqTth(D-B)

[0796] The Taq DN RX HT construct was cut with the enzymes NdeI andBamHI and the larger vector fragment was isolated as detailed above. TheTth DN RX HT construct was also cut with NdeI and BamHI and the smaller(approximately 450 base pairs) Tth fragment was gel isolated andpurified. The Tth NdeI-BamHI insert was ligated into the Taq NdeI-BamHIvector as detailed above to generate the TaqTth(D-B) (DNA sequence SEQID NO:74; amino acid sequence SEQ ID NO:7).

[0797] 6. Construction of TaqTth(Bs-B)

[0798] The Taq DN RX HT construct was cut with the enzymes BstBI andBamHI and the larger vector fragment was isolated as detailed above. TheTth DN RX HT construct was also cut with BstBI and BamHI and the smaller(approximately 633 base pairs) Tth fragment was gel isolated andpurified. The Tth NdeI-BamHI insert was ligated into the Taq NdeI-BamHIvector as detailed above to generate TaqTth(Bs-B) (DNA sequence SEQ IDNO:75; amino acid sequence SEQ ID NO:8).

[0799] 7. Construction of TaqTth(N-Bs)

[0800] The Taq DN RX HT construct was cut with the enzymes NotI andBstBI and the larger vector fragment was isolated as detailed above. TheTth DN RX HT construct was also cut with NotI and BstBI and the smaller(approximately 162 base pairs) Tth fragment was gel isolated andpurified. The Tth NotI-BstBI insert was ligated into the Taq NotI-BstBIvector as detailed above to generate TaqTth(N-Bs) (DNA sequence SEQ IDNO:76; amino acid sequence SEQ ID NO:9).

[0801] 8. Construction of TthTaq(B-S)

[0802] The Tth DN RX HT construct was cut with the enzymes BamHI andSalI and the larger vector fragment was isolated as detailed above. TheTaq DN RX HT construct was also cut with BamHI and SalI and the smaller(approximately 741 base pairs) Tth fragment was gel isolated andpurified. The Taq BamHI-SalI insert was ligated into the Tth BamHI-SalIvector as detailed above to generate the TthTaq(B-S) (DNA sequence SEQID NO:77; amino acid sequence SEQ ID NO:10).

[0803] 9. Construction of Tth Taq(N-B)

[0804] The Tth DN RX HT construct was cut with the enzymes NotI andBamHI and the larger vector fragment was isolated as detailed above. TheTaq DN RX HT construct was also cut with NotI and BamHI and the smaller(approximately 795 base pairs) Tth fragment was gel isolated andpurified. The Taq NotI-BamHI insert was ligated into the Tth NotI-BamHIvector as detailed above to generate the TthTaq(N-B) (DNA sequence SEQID NO:78; amino acid sequence SEQ ID NO:11).

[0805] The cleavage activities of these chimerical proteins werecharacterized as describe in Example 1, part A, and a comparison of thecleavage cycling rates on an RNA target is shown in FIG. 12. As furtherdiscussed in the Description of the Invention, these data show thatelements found in the central third of the TthPol protein are importantin conferring the TthPol-like RNA-dependent cleavage activity on thechimerical proteins comprising portions of TaqPol.

Example 4 Alterations Influencing RNA-Dependent 5′ Nuclease Activity donot Necessarily Influence RNA-Dependent DNA Polymerase Activity

[0806] TthPol is known to have a more active RNA template dependent DNApolymerase than does the TaqPol (Myers and Gelfand, Biochemistry 30:7661[1991]). To determine whether the RNA template dependent 5′ nucleaseactivity of the Thermus DNA Pol I enzymes is related to theirRNA-dependent polymerase activity, the D785N and D787N mutations used tocreate the polymerase-deficient versions of TaqPol and TthPol,respectively were reversed. Polymerase activity was similarly restoredto the TaqTth (N) (DNA sequence SEQ ID NO:79; amino acid sequence SEQ IDNO:12), TaqTth(N-B) (DNA sequence SEQ ID NO:80; amino acid sequence SEQID NO:13), TaqTth(B-S) (DNA sequence SEQ ID NO:81; amino acid sequenceSEQ ID NO:14) chimeras, and the TaqPol(W417L/G418K/E507Q) (DNA sequenceSEQ ID NO:82; amino acid sequence SEQ ID NO:15) mutant proteins.

[0807] Polymerase function was restored in all the above mentionedenzyme mutants by inserting the BamHI to SalI fragment of the native,non-DN sequence into the selected chimera or mutant enzyme. For example,the mutant construct TaqTth(N-B) was cut with the restriction enzymeBamHI (approximate amino acid position 593) and the restriction enzymeSalI (approximate amino acid position 840). The larger vector fragmentwas gel purified as described in Example 3D. The native TaqPol constructwas also cut with the restriction endonucleases BamHI and SalI, and thesmaller insert fragment containing the native amino acid sequence wasalso gel purified. The insert fragment was then ligated into the vectoras detailed in Experimental Example 3D.

[0808] The polymerase activities of these proteins were evaluated byextension of the dT₂₅₋₃₅-oligonucleotide primer with fluorescein-labeleddUTP in the presence of either poly(dA) or poly(A) template. Primerextension reactions were carried out in 10 μl buffer containing 10 mMMOPS, pH 7.5, 5 mM MgSO₄, 100 mM KCl. Forty ng of enzyme were used toextend 10 μM (dT)₂₅₋₃₀ primer in the presence of either 10 μM poly(A)₂₈₆or 1 μM poly(dA)₂₇₃ template, 45 μM dTTP and 5 μM Fl-dUTP at 60° C. for30 min. Reactions were stopped with 10 μl of stop solution (95%formamide, 10 mM EDTA, 0.02% methyl violet dye). Samples (3 μl) werefractionated on a 15% denaturing acrylamide gel and the fraction ofincorporated Fl-dUTP was quantitated using an FMBIO-100 fluorescent gelscanner (Hitachi) equipped with a 505 nm filter as described above.

[0809] As shown in FIG. 16, the DNA-dependent polymerase activities arevery similar for all constructs used in this experiment, whereas theRNA-dependent polymerase activities of TthPol, TaqTth(N) and TaqTth(B-S)are at least 6-fold higher than the activities of TaqPol, TaqTth(N-B)and the TaqPol W417L/G418K/E507Q mutant. From the analysis of theseresults, it can be concluded that the high RNA-dependent DNA polymeraseactivity of TthPol is determined by the C-terminal half of thepolymerase domain (roughly, amino acids 593-830) and that theRNA-dependent 5′ nuclease and polymerase activities are not related toeach other, and are controlled by different regions.

Example 5 Specific Point Mutants in Taq DN RX HT Developed fromInformation from the Chimeric Studies

[0810] The chimeric studies (Example 3, above) suggest that the part ofthe TthPol sequence determining its high RNA-dependent 5′ nucleaseactivity comprises the BstBI-BamHI region located approximately betweenamino acid 382 and 593. Comparison of the amino acid sequences betweenthe BstBI and BamHI regions of Tth DN RX HT and Taq DN RX HT (SEQ IDNOS:165 and 164, respectively) revealed only 25 differences (FIG. 13).Among these, 12 amino acid changes were conservative while 13 of thedifferences resulted in a changes in charge. Since the analysis of thechimeric enzymes suggested that the critical mutations are located inboth the BstBI-NdeI and the NdeI-BamHI regions of Tth DN RX HT, sitespecific mutagenesis was used to introduce the Tth DN RX HT specificamino acids into the BstBI-NdeI and NdeI-BamHI regions of theTaqTth(D-B) and the TaqTth(N-D) respectively.

[0811] Six Tth DN RX HT specific substitutions were generated in theBstBI-NdeI region of the TaqTth(D-B) by single or double amino acidmutagenesis. Similarly, 12 Tth DN RX HT specific amino acid changes wereintroduced at the homologous position of the NdeI-BamHI region of theTaqTth(N-D).

[0812] Plasmid DNA was purified from 200 ml of JM109 overnight cultureusing QIAGEN Plasmid Maxi Kit (QIAGEN, Chatsworth, Calif.) according tothe manufacturer's protocol to obtain enough starting material for allmutagenesis reactions. All site specific mutations were introduced usingthe Transformer Site Directed mutagenesis Kit (Clontech) according tothe manufacturer's protocol; specific sequence information for themutagenic primers used for each site is provided below. One of twodifferent selection primers, Trans Oligo AlwNI/SpeI or Switch OligoSpeI/AlwNI (Clontech, Palo Alto, Calif. catalog #6488-1 or catalog#6373-1) was used for all mutagenesis reactions described. The selectionoligo used in a given reaction is dependent on the restriction sitepresent in the vector. All mutagenic primers were synthesized bystandard synthetic chemistry. Resultant colonies were E.coli strainJM109.

[0813] 1. Construction of TaqTth(D-B) E404H (DNA Sequence SEQ ID NO:83;Amino Acid Sequence SEQ ID NO:16)

[0814] Site specific mutagenesis was performed on pTrc99A TaqTth(D-B)DNA using the mutagenic primer 240-60-01 5′-gag gag gcg ggg cac cgg gccgcc ctt-3′ (SEQ ID NO:277) to introduce the E404H mutation.

[0815] 2. Construction of TaqTth(D-B) F413H/A414R (DNA Sequence SEQ IDNO:84; Amino Acid Sequence SEQ ID NO:17)

[0816] Site specific mutagenesis was performed on pTrc99A TaqTth(D-B)DNA using the mutagenic primer 240-60-02 5′-ctt tcc gag agg ctc cat cggaac ctg tgg ggg agg-3′ (SEQ ID NO:278) to introduce the F413H and theA414R mutations.

[0817] 3. Construction of TaqTth(D-B) W417L/G418K (DNA Sequence SEQ IDNO:85; Amino Acid Sequence SEQ ID NO:18)

[0818] Site specific mutagenesis was performed on pTrc99A TaqTth(D-B)DNA using the mutagenic primer 240-60-03 5′-ctc ttc gcc aac ctg ctt aagagg ctt gag ggg gag-3′ (SEQ ID NO:279) to introduce the W417L and theG418K mutations.

[0819] 4. Construction of TaqTth(D-B) A439R (DNA Sequence SEQ ID NO:86;Amino Acid Sequence SEQ ID NO:19)

[0820] Site specific mutagenesis was performed on pTrc99A TaqTth(ND-B)DNA using the mutagenic primer 240-60-04 5′-agg ccc ctt tcc cgg gtc ctggcc cat-3′ (SEQ ID NO:280) to introduce the A439R mutation.

[0821] 5. Construction of TaqTth(N-D) L451R (DNA Sequence SEQ ID NO:87;Amino Acid Sequence SEQ ID NO:20)

[0822] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-05 5′-acg ggg gtg cgc cgg gac gtg gcctat-3′ (SEQ ID NO:281) to introduce the L415 mutation.

[0823] 6. Construction of TaqTth(N-D) R457Q (DNA Sequence SEQ ID NO:88;Amino Acid Sequence SEQ ID NO:21)

[0824] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-06 5′-gtg gcc tat ctc cag gcc ttg tccctg-3′ (SEQ ID NO:282) to introduce the L415Q mutation.

[0825] 7. Construction of TaqTth(N-D) V463L (DNA Sequence SEQ ID NO:89;Amino Acid Sequence SEQ ID NO:22)

[0826] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-07 5′-ttg tcc ctg gag ctt gcc gag gagatc-3′ (SEQ ID NO:283) to introduce the V463L mutation.

[0827] 8. Construction of TaqTth(N-D) A468R (DNA Sequence SEQ ID NO:90;Amino Acid Sequence SEQ ID NO:23)

[0828] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-08 5′-gcc gag gag atc cgc cgc ctc gaggcc-3′ (SEQ ID NO:284) to introduce the A468R mutation.

[0829] 9. Construction of TaqTth(N-D) A472E (DNA Sequence SEQ ID NO:91;Amino Acid Sequence SEQ ID NO:24)

[0830] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-09 5′-gcc cgc ctc gag gag gag gtc ttccgc-3′ (SEQ ID NO:285) to introduce the A472E mutation.

[0831] 10. Construction of TaqTth(N-D) G499R (DNA Sequence SEQ ID NO:92;Amino Acid Sequence SEQ ID NO:25)

[0832] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-10 5′-ttt gac gag cta agg ctt ccc gccatc-3′ (SEQ ID NO:286) to introduce the G499R mutation.

[0833] 11. Construction of TaqTth(N-D) E507Q (DNA Sequence SEQ ID NO:93;Amino Acid Sequence SEQ ID NO:26)

[0834] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 276-046-04 5′-atc gcc aag acg caa aag acc ggcaag-3′ (SEQ ID NO:287) to introduce the E507Q mutation.

[0835] 12. Construction of TaqTth(N-D) Y535H (DNA Sequence SEQ ID NO:94;Amino Acid Sequence SEQ ID NO:27)

[0836] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-11 5′-aag atc ctg cag cac cgg gag ctcacc-3′ (SEQ ID NO:288) to introduce the Y535H mutation.

[0837] 13. Construction of TaqTth(N-D) S543N (DNA Sequence SEQ ID NO:95;Amino Acid Sequence SEQ ID NO:28)

[0838] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-12 5′-acc aag ctg aag aac acc tac attgac-3′ (SEQ ID NO:289) to introduce the S543N mutation.

[0839] 14. Construction of TaqTth(N-D) 1546V (DNA Sequence SEQ ID NO:96;Amino Acid Sequence SEQ ID NO:29)

[0840] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-13 5′-aag agc acc tac gtg gac ccc ttgccg-3′ (SEQ ID NO:290) to introduce the 1546V mutation.

[0841] 15. Construction of TaqTth(N-D) D551S/I553V (DNA Sequence SEQ IDNO:97; Amino Acid Sequence SEQ ID NO:30)

[0842] Site specific mutagenesis was performed on pTrc99AtaqTth(N-D) DNAusing the mutagenic primer 240-60-14 5′-att gac ccc ttg ccg agc ctc gtccac ccc agg acg ggc-3′ (SEQ ID NO:291) to introduce the D551S and the1553V mutations.

[0843] 16. Construction of TaqDN RX HT W417L/G418K1E507Q (DNA SequenceSEQ ID NO:98; Amino Acid Sequence SEQ ID NO:31)

[0844] The TaqDN RX HT W417L/G418K/E507Q triple mutant was made bycombining the TaqTth(D-B)W417L/G418K with the TaqTth(N-D) E507Q.TaqTth(D-B)W417L/G418K was cut with the restriction enzymes NdeI andBamHI, and the larger, vector fragment was isolated as detailed inExample 3. The TaqTth(N-D) E507Q construct was also cut with NdeI andBamHI and the smaller (approximately 795 base pairs) fragment was gelisolated and purified as detailed in Example 3. The NdeI-BamHI insertwas ligated into the gel purified vector, as detailed in Example 3.

[0845] 17. Construction of TaqDN RX HT W417L/E507Q (DNA Sequence SEQ IDNO:99; Amino Acid Sequence SEQ ID NO:32)

[0846] Starting with TaqDN RX HT W417L/G418K/E507Q described above,mutagenic primer 337-01-02: 5′-TTC GCC AAC CTG CTT GGG AGG CTT GAG GGGGAG-3′ (SEQ ID NO:292) was used in a site specific mutagenesis reactionto change the K at amino acid position 418 back to the wild-type aminoacid, G. Site specific mutagenesis was done using the Transformer SiteDirected Mutagenesis Kit (Clonetech) according to the manufacturer'sinstructions, and as described in Experimental Example 4.

[0847] 18. Construction of TaqDN RX HT G418K/E507Q (DNA Sequence SEQ IDNO:100; Amino Acid Sequence SEQ ID NO:33)

[0848] Starting with TaqDN RX HT W417L/G418K/E507Q described above,mutagenic primer 337-01-01: 5′-CTC TTC GCC AAC CTG TGG AAG AGG CTT GAGGGG-3′ (SEQ ID NO:293) was used in a site specific mutagenesis reactionto change the L at amino acid position 417 back to the wild-type aminoacid, W. Site specific mutagenesis was done using the Transformer SiteDirected Mutagenesis Kit (Clonetech) according to the manufacturer'sinstructions, and as described in Experimental Example 4.

[0849] Expression and purification of mutant proteins was done asdetailed in Example 3, and the cleavage activities of these proteinswere characterized as describe in Example 1, part A. A comparison of thecleavage cycling rates of a selection of these mutant proteins on an RNAtarget is shown in FIG. 14. As further discussed in the Description ofthe Invention, these data show that amino acids in the regions 417/418and amino acid 507 are important in the conferring the TthPol-likeRNA-dependent cleavage activity on the chimerical proteins comprisingportions of TaqPol in combination with portions of TthPol that are notindependently capable of providing enhanced RNA dependent activity(i.e., the D-B and N-D portions of Tth). As described in the Descriptionof the Invention, Taq DN RX HT variant carrying only the W417L, G418Kand E507Q substitutions were created. By comparing their cleavage ratesto that of Tth DN RX HT on the IL-6 RNA substrate as described inExample 1, these mutations were determined to be sufficient to increasethe Taq DN RX HT activity to the Tth DN RX HT level. FIG. 15 shows thatthe Taq DN RX HT W417L/G418K/E507Q and Taq DN RX HT G418K/E507Q mutantshave 1.4 times higher activity than Tth DN RX HT and more than 4 foldhigher activity than Taq DN RX HT, whereas the Taq DN RX HT W417L/E507Qmutant has the same activity as the enzyme, which is about 3 fold higherthan Taq DN RX HT. These results demonstrate that K418 and Q507 ofTthPol are particularly important amino acids in providing RNA dependent5′ nuclease activity that is enhanced compared to TaqPol.

Example 6 RNA-Dependent 5′ Nuclease Properties of the Taq DN RX HTG418K/E507Q 5′ Nuclease are Similar to Tth DN RX HT with Respect to Saltand Temperature Optima

[0850] To determine if the G418K/E507Q mutations caused any significantchanges in the properties of the Taq DN RX HT mutant in addition to theincreased cleavage rate with the RNA target, the Taq DN RX HTG418K/E507Q (SEQ ID NO:33), Taq DN RX HT (SEQ ID NO₂₇₆), and Tth DN RXHT (SEQ ID NO:274) enzymes were compared in the RNA template dependent5′ nuclease assay under conditions where temperature and theconcentrations of salt and divalent ions were varied. The upstream DNAand the template RNA strands of the substrate used in this study werelinked into a single IrT molecule (SEQ ID NO:166) as shown in FIG. 20A,and the labeled downstream probe (SEQ ID NO:167) was present in largeexcess. The 5′ end of the target RNA strand was blocked with abiotin-streptavidin complex to prevent any non-specific degradation bythe enzyme during the reaction (Lyamichev et al., Science 260:778[1993], Johnson et al., Science 269:238 [1995]). The cleavage rates forTaq DN RX HT G418K/E507Q, Taq DN RX HT, and Tth DN RX HT are plotted asfunctions of temperature in FIG. 20B. The closed circles representenzyme Taq DN RX HT, the open circles represent enzyme Tth DN RX HT, andthe Xs represent enzyme Taq DN RX HT G418K/E507Q. The difference in theactivities of Tth and Taq DN RX HT enzymes with the IrT substrate iseven greater than the difference found with the IL-6 RNA substrate whentested in a cleavage assay as described in Example 1. The G418K/E507Qmutations increase the activity of the Taq enzyme more than tenfold andby 25% compared with the Tth enzyme. All three enzymes show a typicaltemperature profile of the invasive signal amplification reaction andhave the same optimal temperature. No significant effect of G418K/E507Qmutations on DNA dependent 5′ nuclease activity of Taq DN RX HT with theall-DNA substrate analogous to IrT (SEQ ID NO:168) under the sameconditions was found.

[0851] The effects of KCl and MgSO₄ concentrations on the 5′ nucleaseactivity of Taq DN RX HT G418K/E507Q, Taq DN RX HT, and Tth DN RX HTwith the IrT substrate are shown in FIG. 20C and D. The activities ofall enzymes have similar salt dependencies with an optimal KClconcentration of 100 mM for Taq DN RX HT G418K/E507Q and Tth DN RX HTand 50 mM for Taq DN RX HT. The optimal MgSO₄ concentration for allenzymes is approximately 8 mM. The analysis of the data presented inFIG. 20 suggests that the properties of Taq DN RX HT G418K/E507Q aremuch closer to those of Tth DN RX HT rather than Taq DN RX HT confirmingthe key role of the G418K/E507Q mutations in the recognition of thesubstrate with an RNA target.

[0852] To understand the mechanism of the reduction of the 5′ nucleaseactivity in the presence of an RNA versus a DNA target, the Michaelisconstant (K_(m)) and the maximal catalytic rate (k_(cat)) of all threeenzymes were determined, using an excess of the IrT substrate (SEQ IDNO:166) and the downstream probe (SEQ ID NO:167) and a limiting enzymeconcentration. For these measurements, ten-μl reactions were assembledcontaining 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05% Nonidet P-40, 10μg/ml tRNA, 4 mM MgCl₂, 1 nM of enzyme (Taq DN RX HT, Tth DN RX HT, orTaq DN RX HT G418K/E507Q) and different concentrations (0.125, 0.25, 0.5or 1 μM) of an equimolar mixture of the IrT target and the downstreamprobe. The cleavage kinetics for each enzyme and each substrateconcentration were measured at 46° C. Reactions were stopped by theaddition of 10 μl of 95% formamide containing 10 mM EDTA and 0.02%methyl violet (Sigma). One μl of each stopped reaction digest wasfractionated on a 20% denaturing acrylamide gel (19:1 cross-linked),with 7M urea, and in a buffer of 45 mM Tris-borate, pH 8.3, 1.4 mM EDTA.Gels were scanned on an FMBIO-100 fluorescent gel scanner (Hitachi)using a 585 nm filter. The fraction of cleaved product (determined fromintensities of bands corresponding to uncut and cut substrate with FMBIOAnalysis software, version 6.0, Hitachi) was plotted as a function ofreaction time. The initial cleavage rates were determined from theslopes of linear part of the cleavage kinetics and were defined as theconcentration of cut product divided by the enzyme concentration and thetime of the reaction (in minutes). The Michaelis constant K_(m) and themaximal catalytic rate k_(cat) of each enzyme with IrT substrate weredetermined from the plots of the initial cleavage rate as functions ofthe substrate concentration.

[0853] It was found that all three enzymes have similar K_(m) values (inthe range of 200-300 nM) and k_(cat) values of approximately 4 min⁻¹ forTaq DN RX HT and Tth DN RX HT and of 9 min⁻¹ for Taq DN RX HTG418K/E507Q. That the G418K/E507Q mutations increase the k_(cat) of TaqDN RX HT more than two fold, but have little effect on K_(m) suggestthat the mutations position the substrate in an orientation moreappropriate for cleavage, rather than simply increase the bindingconstant.

Example 7 Use of Molecular Modeling to Further Improve RNA-Dependent 5′Nuclease Activity

[0854] A. Point Mutants

[0855] To develop enzymes with altered function, sequence changes wereintroduced by site-specific mutagenesis in predetermined locations or byrandom mutagenesis. Locations for site-specific mutagenesis were chosenbased on evidence from chimeric studies, relevant published literature,and molecular modeling. Seven additional mutant enzymes were developedfrom the Tth DN RX HT enzyme, and twenty additional mutant enzymes weredeveloped from the Taq DN RX HT enzyme, both discussed previously. Someof the mutant enzymes are the result of multiple mutagenesis reactions,that is, more than one change has been introduced to obtain the finalproduct. Mutation reactions were done using the Tth DN RX HT construct(SEQ ID NO:273) described in Example 2C2, or the Taq DN RX HT construct(SEQ ID NO:275), described in Example 2C1 unless otherwise stated.Plasmid DNA was purified from 200 ml of JM109 overnight culture usingQIAGEN Plasmid Maxi Kit (QIAGEN, Chatsworth, Calif.) according to themanufacturer's protocol to obtain enough starting material for allmutagenesis reactions. All site-specific mutations were introduced usingthe Transformer Site Directed mutagenesis Kit (Clontech) according tothe manufacturer's protocol. One of two different selection primers,Trans Oligo AlwNI/SpeI or Switch Oligo SpeI/AlwNI (Clontech, Palo AltoCalif. catalog #6488-1 or catalog #6373-1) was used for all mutagenesisreactions described. The selection oligo used in a given reaction isdependent on the restriction site present in the vector. All mutagenicprimers for both the site-specific mutagenesis and the randommutagenesis were synthesized by standard synthetic chemistry. Resultantcolonies for both types of reactions were E. coli strain JM109. Randommutagenesis methods are described below.

[0856] Mutants were tested via the rapid screening protocol detailed inExample 1. Then, if more detailed analysis was desired, or if a largerprotein preparation was required, expression and purification of mutantproteins was done as detailed in Example 3.

[0857] 1. Construction of Tth DN RX HT H641A, Tth DN RX HT H748A, Tth DNRX HT H786A

[0858] Site specific mutagenesis was performed on pTrc99A Tth DN RX HTDNA using the mutagenic primer 583-001-02: 5′-gct tgc ggt ctg ggt ggcgat gtc ctt ccc ctc-3′ (SEQ ID NO:294) to introduce the H641A mutation(DNA sequence SEQ ID NO:101; amino acid sequence SEQ ID NO:34), or themutagenic primer 583-001-03: 5′ cat gtt gaa ggc cat ggc ctc cgc ggc ctccct-3′ (SEQ ID NO:295) to generate the H748A mutant (DNA sequence SEQ IDNO:102; amino acid sequence SEQ ID NO:35), or the mutagenic primer583-001-04: 5′-cag gag gag ctc gtt ggc gac ctg gag gag-3′ (SEQ IDNO:296) to generate the H786A mutant enzyme (DNA sequence SEQ ID NO:103;amino acid sequence SEQ ID NO:36).

[0859] 2. Construction of Tth DN RX HT (H786A/G506K/Q509K)

[0860] Starting with the mutant Tth DN RX HT H786A, generated above,site specific mutagenesis was done using the mutagenic primer604-022-02: 5′-gga gcg ctt gcc tgt ctt ctt cgt ctt ctt caa ggc ggg aggcct-3′ (SEQ ID NO:297) to generate this variant termed “TthAKK”, (DNAsequence SEQ ID NO:104; amino acid sequence SEQ ID NO:37).

[0861] 3. Construction of Taq DN RX HT (W417L/G418K/E507Q/H784A)

[0862] Mutagenic oligonucleotide 158-029-02: 5′-gag gac cag ctc gtt ggcgac ctg aag gag cat-3′ (SEQ ID NO:298) was used in a site specificmutagenesis reaction to introduce the H784A mutation and generate thisconstruct termed “Taq4M” (DNA sequence SEQ ID NO:105; amino acidsequence SEQ ID NO:38).

[0863] 4. Construction of Taq4M H639A, Taq4M R587A, Taq4M G504K andTaq4M G80E

[0864] Site specific mutagenesis was done on the Taq4M mutant, usingprimer 473-010-11: 5′-gaggggcgggacatcgccacggagaccgccagc-3′ (SEQ IDNO:299) to generate the Taq 4M H639A mutant (DNA sequence SEQ ID NO:106;amino acid sequence SEQ ID NO:39), primer 473-010-10: 5′-cag aac atc cccgtc gcc acc ccg ctt ggg cag-3′ (SEQ ID NO:300) to generate Taq 4M R587A(DNA sequence SEQ ID NO:107; amino acid sequence SEQ ID NO:40), primer300-081-06: 5′-ggg ctt ccc gcc atc aag aag acg gag aag acc-3′ (SEQ IDNO:301) to generate Taq 4M G504K (DNA sequence SEQ ID NO:108; amino acidsequence SEQ ID NO:41), and primer 330-088-04: 5′-cta ggg ctt ccc gccatc aag aag acg caa aag acc ggc-3′ (SEQ ID NO:302) to generate the Taq4M G80E mutant (DNA sequence SEQ ID NO:109; amino acid sequence SEQ IDNO:42).

[0865] 5. Construction of Taq 4M P88E/P90E and Taq 4M L109F/A110T

[0866] Starting with Taq 4M described above, site specific mutagenesiswas done using primer 473-087-03: 5′-ccg ggg aaa gtc ctc ctc cgt ctc ggcccg gcc cgc ctt-3′ (SEQ ID NO:303) to generate the P88E/P90E mutations(DNA sequence SEQ ID NO:110; amino acid sequence SEQ ID NO:43), orprimer 473-087-05: 5′-cgg gac ctc gag gcg cgt gaa ccc cag gag gtc cac-3′(SEQ ID NO:304) to generate the L109F/A110T mutations (DNA sequence SEQID NO:111; amino acid sequence SEQ ID NO:44).

[0867] 6. Construction of Taq DN RX HT(W417L/G418K/G499R/A502K/I503L/G504K/E507K/H784A)

[0868] Two PCR reactions were performed, first using construct Taq4M(Taq W417L/G418K/G504K/E507Q/H784A) as a template. Using primers158-84-01 5′-CTCCTCCACGAGTTCGGC-3′ (SEQ ID NO:305) and 535-33-02 5′-ACCGGT CTT CTT CGT CTT CTT CAA CTT GGG AAG CCT GAG CTC GTC AAA-3′ (SEQ IDNO:306) a 620 base pair PCR fragment was generated. Another 510 basepair PCR product was generated using primer 535-33-01 5′-AAG ACG AAG AAGACC GGT AAG CGC TCC ACC AGC-3′ (SEQ ID NO:307) and 330-06-03 5′-GTC GACTCT AGA TCA GTG GTG GTG GTG GTG GTG CTT GGC CGC CCG GCG CAT C-3′ (SEQ IDNO:308). The two PCR products overlap such that a final recombinant PCRamplification was done using the outside primers 158-84-01 and 330-06-03to yield the 1182 base pair product. The recombinant PCR product wasdigested with the restriction enzymes NotI and BamHI according to themanufacturer's instructions to yield a 793 base pair fragment. Theparent plasmid Taq4M was also digested with the same enzymes and used asthe vector for ligation. All DNA fragments were TAE agarose gel purifiedprior to ligation. The fragment was ligated into the vector, andtransformed into JM109 cells, thus incorporating the mutations G499R,A502K, 1503L, and E507K as well as the restriction endonuclease site,AgeI. This construct is termed “Taq 8M” (DNA sequence SEQ ID NO:112;amino acid sequence SEQ ID NO:45).

[0869] B. Random Mutagenesis

[0870] Numerous enzymes with altered function were generated via randommutagenesis. The regions of the protein targeted for random mutagenesiswere chosen based on molecular modeling data and from information in theliterature. Different mutagenic primers were used to introduce mutationsinto different regions of the protein. Random mutagenesis was performedon the Taq variant Taq 4M G504K (Taq DN RX HTW417L/G418K/G504K/E507Q/H784A/) (SEQ ID NO:108) described above andmutant PCR fragments generated in the mutagenesis reaction wereexchanged for homologous regions in Taq8M (SEQ ID NO:112) unlessotherwise stated.

[0871] Random mutagenesis was also performed on the Tth DN RX HT H786A(SEQ ID NO:103) described above. Mutant PCR fragments generated with theTth DN RX HT H786A template were exchanged for homologous regions in theunaltered Tth DN RX HT H786A.

[0872] 1. Random Mutants in Amino Acid Residues 500-507 or 513-520

[0873] The first mutagenic oligonucleotide, 535-054-01: 5′-gga gcg cttacc ggt ctt (ttg cgt ctt ctt gat ctt ggg aag) cct tag ctc gtc aaa gag-3′(SEQ ID NO:309) was used in conjunction with 158-84-01: 5′-CTC CTC CACGAG TTC GGC-3′ (SEQ ID NO:310) to install random residues from aminoacid position 500 to 507 of Taq polymerase variant Taq DN RX HTW417L/G418K/G504K/E507Q/H784A (SEQ ID NO:108). This was accomplished bysynthesizing the primer 535-054-01 such that only 91% of the baseswithin the parenthesis are unaltered while the remaining 9% of the basesare an equal mixture of the other 3 nucleotides. The initial, unalteredsequence of this oligo includes the G499R, A502K and the Q507K changes.

[0874] To generate mutations in the region 500-507, primer 535-054-01and primer 158-84-01 were used in a PCR reaction, using the AdvantagecDNA PCR kit (Clonetech) and Taq variant described above, as the target.This PCR fragments was then run on a 1% TAE agarose gel, excised andpurified with QlAquick Gel Extraction Kit (Qiagen, Valencia Calif.,catalog # 28706). The purified fragment was cut with NotI and AgeI andligated into pTaq8M that had been linearized with NotI and AgeI. JM109E.coli cells (Promega) were transformed with the ligated products.Clones were tested as described below.

[0875] The second mutagenic oligonucleotide (used in a separatereaction) 535-054-02: 5′-caa aag acc ggt aag cgc (tcc acc agc gcc gccgtc ctg gag) gcc ctc cgc gag gcc cac-3′ (SEQ ID NO:311) was used inconjunction with 330-06-03: 5′-GTC GAC TCT AGA TCA GTG GTG GTG GTG GTGGTG CTT GGC CGC CCG GCG CAT C-3′ (SEQ ID NO:312) to install randomresidues from amino acid 513-520. The bases within the parenthesis ofprimer 535-054-02 are also 91% wild-type and 3% each of the other 3nucleotides.

[0876] To generate mutations in the region 513-520, primer 535-054-02and primer 535-054-02 were used in a PCR reaction with Taq DN RX HTW417L/G418K/G504K/E507Q/H784A (SEQ ID NO:108) as template, as describedabove. The resulting PCR fragment was purified as above and cut with therestriction enzymes AgeI and BamHI. The cut fragment was then ligatedinto the Taq8M construct, also linearized with AgeI and BamHI. JM109E.coli cells were transformed with the ligated products. Clones weretested as described Example 1. Mutants developed from these include:

[0877] Taq DN RX HT W417L/G418K/G499R/A502K/K504N/E507K/H784A (MI-13)(DNA sequence SEQ ID NO:113; amino acid sequence SEQ ID NO:46).

[0878] Taq DN RX HT W417L/G418K/G499R/L500I/A502K/G504K/Q507H/H784A(M1-36) (DNA sequence SEQ ID NO:114; amino acid sequence SEQ ID NO:47).

[0879] Taq DN RX HTW417L/G418K/G499RJA502K/I503L/G504K/E507K/T514S/H784A (M2-24) (DNAsequence SEQ ID NO:115; amino acid sequence SEQ ID NO:48).

[0880] Taq DN RX HTW417L/G418K/G499R/A502K/I503L/G504K/E507K/V518L/H784A (M2-06) (DNAsequence SEQ ID NO:116; amino acid sequence SEQ ID NO:49).

[0881] 2. TthDN RX HT H786A Random Mutagenesis

[0882] To generate mutants in the helix-hairpin-helix region of theTthDN RX HT H786A (SEQ ID NO:36) enzyme, two different PCR reactionswere performed using the H786A (SEQ ID NO:103) mutant as a template. Thetwo PCR products overlap such that a recombinant PCR reaction can beperformed (Higuchi, in PCR Technology, H. A. Erlich, ed., StocktonPress, New York. pp61-70 [1989]). This final PCR product is thenexchanged with the homologous region of the TthDN H786A mutant by usingrestriction enzyme sites located on the ends of the fragment and withinthe TthDN H786A sequence.

[0883] Starting with TthDN H786A discussed above, and using primer604-08-06: 5′-gtc gga ggg gtc ccc cac gag-3′ (SEQ ID NO:313) and primer390-76-08: 5′-tgt gga att gtg agc gg (SEQ ID NO:314), a 620 base pairPCR fragment was generated. PCR reactions were performed using theAdvantage cDNA PCR kit (Clontech) according to manufacturer'sinstructions. This PCR product includes amino acids 1-194. No mutationswere introduced via this reaction, however the restriction enzyme siteEcoRI is present at the 5′ end.

[0884] Starting with TthDN RX HT H786A discussed above, and usingmutagenic primer 604-08-05: 5′-ctc gtg ggg gac ccc tcc gac aac ctc (cccggg gtc aag ggc atc ggg gag aag acc gcc) ctc aag ctt ctc aag-3′ (SEQ IDNO:315) and primer 209-74-02: 5′-gtg gcc tcc ata tgg gcc agg α-3′ (SEQID NO:316) a 787 base pair PCR fragment was generated. PCR reactionswere done as above. This fragment does contain random mutations, due tothe presence of the mutagenic primer, 604-08-05. The bases within theparenthesis of this primer were synthesized such that 91% of thesequence is wild-type, while the additional 9% is evenly divided betweenthe remaining 3 bases.

[0885] The two PCR fragments overlap, and were combined in a recombinantPCR reaction. Primers 390-76-08 and 209-74-02 were added, and theAdvantage cDNA PCR kit (Clontech) was again used according tomanufacturer's instructions. A 1380 base pair product was generated fromthis reaction.

[0886] The recombinant PCR product was cut with the restriction enzymesEcoRI and NotI according to the manufacturer's instructions to yield a986 base pair fragment. TthDN RX HT H786A was prepared by cutting withthe same enzymes. The fragment was then ligated into the vector, andtransformed into JM109 cells. New mutants developed from this set ofreactions include:

[0887] TthDN RX HT H786A/P197R/K200R (DNA sequence SEQ ID NO:117; aminoacid sequence SEQ ID NO:50).

[0888] TthDN RX HT H786A/K205Y (DNA sequence SEQ ID NO:118; amino acidsequence SEQ ID NO:51).

[0889] TthDN RX HT H786A/G203R (DNA sequence SEQ ID NO:119; amino acidsequence SEQ ID NO:52).

[0890] 3. Construction of Taq DN RX HT W417L/G418K/H784AL109F/A110T/G499R/A502K/I503L/G504K/E507K/T514S (Taq SS)

[0891] Starting with Taq DN RX HTW417L/G418K/G499R/A502K/I503L/G504K/E507K/T514S/H784A (SEQ ID NO:115)mutant described above, primer 473-087-05: 5′-cgg gac ctc gag gcg cgtgaa ccc cag gag gtc cac-3′ (SEQ ID NO:317) was used in conjunction withthe appropriate selection primer in a site specific mutagenesis reactionto incorporate the L109F and A110T mutations to generate this enzyme,termed “TaqSS” (DNA sequence SEQ ID NO:120; amino acid sequence SEQ IDNO:53).

[0892] 4. Construction of Taq DN RX HT W417L/G418L/H784AP88E/P90E/G499R/A502K/I503L/G504K/E507K/T514S

[0893] Starting with Taq DN RX HTW417L/G418K/G499R/A502K/I503L/G504K/E507K/T514S/H784A (SEQ ID NO:115)mutant described above, primer 473-087-03: 5′-ccg ggg aaa gtc ctc ctccgt ctc ggc ccg gcc cgc ctt-3′ (SEQ ID NO:318) was used in conjunctionwith the appropriate selection primer in a site specific mutagenesisreaction to incorporate the P88E and P90E mutations to generate thisenzyme (DNA sequence SEQ ID NO:121; amino acid sequence SEQ ID NO:54).

[0894] 5. TaqSS Random Mutagenesis

[0895] Random mutagenesis was used to introduce additional changes inthe helix-hairpin-helix domain of the TaqSS mutant (SEQ ID NO:120). Themutagenesis was done as described in example 9 above. In the first step,two different but overlapping PCR products were generated. One of thePCR products, generated with oligos 390-76-08 (SEQ ID NO:314), and604-08-04: 5′-gtc gga ctc gtc acc ggt cag ggc-3′ (SEQ ID NO:319)incorporates the EcoRI site into the fragment, but does not incorporateany mutations. The second PCR product utilizes mutagenic primer604-08-03: 5′-ctg acc ggt gac gag tcc gac aac ctt (ccc ggg gtc aag ggcatc ggg gag aag acg gcg) agg aag ctt ctg gag-3′ (SEQ ID NO:320) andprimer 209-74-02 (SEQ ID NO:316). This fragment contains random pointmutations, and when combined via recombinant PCR with the firstfragment, can be cut with the restriction enzymes EcoRI and NotI, andligated into the TaqSS construct, also cut with EcoRI and NotI. Theligated construct was then transformed into JM109. Colonies werescreened as described below. Enzymes developed from this mutagenesisinclude:

[0896] TaqSS K198N (DNA sequence SEQ ID NO:112; amino acid sequence SEQID NO:55).

[0897] TaqSS A205Q (DNA sequence SEQ ID NO:123; amino acid sequence SEQID NO:56).

[0898] TaqSS 1200M/A205G (DNA sequence SEQ ID NO:124; amino acidsequence SEQ ID NO:57).

[0899] TaqSS K203N (DNA sequence SEQ ID NO:125; amino acid sequence SEQID NO:58).

[0900] TaqSS T204P (DNA sequence SEQ ID NO:126; amino acid sequence SEQID NO:59).

[0901] 6. Construction of TaqSS R677A

[0902] To generate enzymes with sequence changes in both the arch regionand in the polymerase region, additional specific point mutations weregenerated in TaqSS. Site specific mutagenesis was performed as describedabove using the oligo 473-060-10: 5′-tag ctc ctg gga gag ggc gtg ggc cgacat gcc-3′ (SEQ ID NO:321) to generate the TaqSS R677A mutant (DNAsequence SEQ ID NO:127; amino acid sequence SEQ ID NO:60).

[0903] 7. Construction of TaqTthAKK (DNA Sequence SEQ ID NO:128; AminoAcid Sequence SEQ ID NO:61) and TthTaq5M (DNA Sequence SEQ ID NO:129;Amino Acid Sequence SEQ ID NO:62)

[0904] Chimeric mutant TaqTthAKK and TthTaq5M were generated by cuttingTth DN RX HT (H786A/G506K/Q509K) (SEQ ID NO:104; here abbreviatedTthAKK) or Taq 4M G504 (SEQ ID NO:108; here abbreviated Taq 5M) with therestriction endonucleases EcoRI and NotI. The smaller insert fragmentsas well as the larger vector fragments were gel purified as detailed inExample 3D, and the insert fragments were exchanged between the twomutants and ligated as described in Example 3D. Screening andverification of the construct sequence was also done as in Example 3D.

Example 8 Improvement of RNA-Dependent 5′ Nuclease Activity in OtherPolymerases

[0905] Information gained from the TaqPol/TthPol recombinations,mutagenesis and modeling, was used to make comparable mutations inadditional DNA polymerases and examined the effects on the cleavageactivities of these enzymes. The DNA polymerases of Thermus filiformus(TfiPol) and Thermus scotoductus (TscPol) were cloned and purified asdescribed in Example 2. The mutagenesis of these two proteins isdescribed below.

[0906] A. Construction of TfilPolDN2M

[0907] Mutagenesis of pTrc99a-TfiPol (SEQ ID NO:249) was done using theQuikChange site-directed mutagenesis kit (Stratagene) according to themanufacturer's protocol. The P420K mutation was made with the followingtwo oligonucleotides; 5′-CTTCCAGAACCTCTTTAAACGGCTTTCCGAGAAG (SEQ IDNO:322) and 5′-CTTCTCGGAAAGCCGTTTAAAGAGGTTCTGGAAG (SEQ ID NO:323). TheE507Q mutation was made with the following two oligonucleotides;5′-CCGGTGGGCCGGACGCAGAAGACGGGCAAGC (SEQ ID NO:324) and5′-GCTTGCCCGTCTTCTGCGTCCGGCCCACCGG (SEQ ID NO:325). The D785N mutationwas made with the following two oligonucleotides;5′-CTCCTCCAAGTGCACAACGAGCTGGTCCTGG (SEQ ID NO:326) and5′-CCAGGACCAGCTCGTTGTGCACTTGGAGGAG (SEQ ID NO:327). The plasmidcontaining all three mutations is called pTrc99a-TfiPolDN2M, (DNAsequence SEQ ID NO:130; amino acid sequence SEQ ID NO:63).

[0908] B. Construction of TscPolDN2M

[0909] Mutagenesis of pTrc99a-TscPol (SEQ ID NO:253) was done with theQuikChange site-directed mutagenesis kit (Stratagene) according to themanufacturer's protocol. The E416K mutation was made with the followingtwo oligonucleotides; 5′-GCCGCCCTCCTGAAGCGGCTTAAGGG (SEQ ID NO:328) and5′-CCCTTAAGCCGCTTCAGGAGGGCGGC (SEQ ID NO:329). The E505Q mutation wasmade with the following two oligonucleotides;5′-ATCGGCAAGACGCAGAAGACGGGCAAGC (SEQ ID NO:330) and5′-GCTTGCCCGTCTTCTGCGTCTTGCCGAT (SEQ ID NO:331). The D783N mutation wasmade with the following two oligonucleotides;5′-TTGCAGGTGCACAACGAACTGGTCCTC (SEQ ID NO:332) and5′-GAGGACCAGTTCGTTGTGCACCTGCAA (SEQ ID NO:333). The plasmid containingall three mutations is called pTrc99a-TscPolDN2M, (DNA sequence SEQ IDNO:131; amino acid sequence SEQ ID NO:64).

[0910] C. Chimerics of Tsc, Tfi, Tth and Taq Mutants

[0911] 1. Construction of TfiTth AKK (DNA Sequence SEQ ID NO:132; AminoAcid Sequence SEQ ID NO:65), TscTthAKK (DNA Sequence SEQ ID NO:133;Amino Acid Sequence SEQ ID NO:66), TfiTaq5M (DNA Sequence SEQ ID NO:134;Amino Acid Sequence SEQ ID NO:67) and TscTaq5M (DNA Sequence SEQ IDNO:135; Amino Acid Sequence SEQ ID NO:68)

[0912] To generate chimeric enzymes between Tth DN RX HT(H86A/G506K/Q509K) (here abbreviated TthAKK, SEQ ID NO:104) or Taq 4MG504 (here abbreviated Taq 5M, SEQ ID NO:108), and Tfi DN 2M (SEQ IDNO:130), or Tsc DN 2M (SEQ ID NO:131), additional restrictionendonuclease sites were introduced by site specific mutagenesis into thenamed Tfi and Tsc mutants. Mutagenic primers 700-011-01 5′-cag acc atgaat tcc acc cca ctt ttt gac ctg gag-3′ (SEQ ID NO:334) and 700-011-025′-gtg gac gcg gcc gcc cga ggc cgc cgc cag ggc cag-3′ (SEQ ID NO:335)were used to introduce an EcoRI site at amino acid position 1 and a NotIsite at amino acid position 331 in Tfi DN 2M. Mutagenic primers700-011-03 5′-cag acc atg aat tcc ctg ccc ctc ttt gag ccc aag-3′ (SEQ IDNO:336) and 700-011-04 5′-gta aac cgc gcc gcc cca ggc ggc ggc caa ggcgtt-3′ (SEQ ID NO:337) were used to introduce an EcoRI site at aminoacid position 1 and a NotI site at amino acid position 327 in Tsc DN 2M.PCR reactions were done using the Advance cDNA PCR kit (Clonetech)according to manufacturer's instructions and either Tfi DN 2M or Tsc DN2M as target, with their corresponding primers. The 1017 base pair PCRproducts were cut with both EcoRI and NotI to yield 993 base pair insertfragments that were gel purified as described in Example 3D. The mutantsTaq4M G504K (SEQ ID NO:108) and Tth DN RX HT (H786A/G506K/Q509K) (SEQ IDNO:104) were also cut with EcoRI and NotI, and the larger, vectorfragment was gel isolated as above. Ligations were performed as detailedin Example 3D, as was the screening and verification of the newconstructs.

Example 9 Additional Enzymes Having Improved RNA-Dependent 5′ NucleaseActivity

[0913] Generation of Tfi DN 2M(AN)

[0914] To facilitate later cloning steps, an endogenous restrictionenzyme site (Not I) was removed from the polymerase region of theTfiPolDN2M gene (SEQ ID NO:130 described in Example 8A), and a uniqueNot I site was inserted in a more advantageous position.

[0915] The endogeneous Not I site was removed as follows. The QUIKCHANGESite-Directed Mutagenesis Kit from (Stratagene) was used according tomanufacturer's instructions with the mutagenic primers5′-gag-gtg-gag-cgg-ccc-ctc-tcc-cgg-gtc-ttg (SEQ ID NO: 338) and5′-caa-gac-ccg-gga-gag-ggg-ccg-ctc-cac-ctc (SEQ ID NO: 339). The newconstruct was named Tfi DN 2M(ΔN) (DNA sequence SEQ ID NO: 340; aminoacid sequence SEQ ID NO: 341).

[0916] Generation of Tfi DN 2M(N), Tse DN 2M(N)

[0917] To install a unique NotI site (at amino acid position 328) in TfiDN 2M(AN) (SEQ ID NO: 340), primers 886-088-07 (SEQ ID NO: 342)5′-tgg-cgg-cgg-cct-cgg-gcg-gcc-gcg-tcc-acc-ggg-caa-ac-3′ and 700-010-035′-ctt-ctc-tca-tcc-gcc-aaa-aca-gcc (SEQ ID NO: 343) were used in a PCRreaction with Tfi DN 2M(AN) (SEQ ID NO: 340) as template. The resultingPCR fragment was purified and cut with the restriction enzymes NotI andSalI. The cut fragment was then ligated into the TfiTthAKK (SEQ IDNO:132, described in example 8,C) construct which was also digested withNotI and SalI. The new construct was termed Tfi DN 2M(N) (DNA sequenceSEQ ID NO: 345; amino acid sequence SEQ ID NO: 346).

[0918] To introduce a Not I restriction endonuclease site into mutantTscPol DN 2M (previously described in Example 8B above), PCR wasperformed with primers: 886-088-05 (SEQ ID NO: 344)5′-tgg-ccg-ccg-cct-ggg-gcg-gcc-gcg-ttt-acc-ggg-cgg-ag-3′ and 700-010-03(SEQ ID NO: 343) 5′-ctt-ctc-tca-tcc-gcc-aaa-aca-gcc-3′ using TscPolDN2M(SEQ ID NO:131) as template. The NotI-SalI digested fragment of the PCRproduct was then sub-cloned into NotI and SalI digested TscTthAKK vector(SEQ ID NO:133). The resulting construct was termed Tsc DN 2M(N) (DNAsequence SEQ ID NO:347; amino acid sequence SEQ ID NO:348).

[0919] Generation of Tfi DN 2M(N)AKK and Tsc DN2M(N)AKK

[0920] To generate the “AKK” set of mutations (G504K/E507K/H784A/D785N),site specific mutagenesis was done on the Tfi DN 2M(N) mutant (DNAsequence SEQ ID NO: 345), using primers 959-022-01 to −04:5′-ggc-ctc-acc-ccg-gtg-aag-cgg-acg-aag-aag-acg-ggc-aag-cgc-3′,5′-gcg-ctt-gec-cgt-ctt-ctt-cgt-ccg-ctt-cac-cgg-ggt-gag-gcc-3′,5′-ctc-ctc-ctc-caa-gtg-gcc-aac-gag-ctg-gtc-ctg-3′,5′-cag-gac-cag-ctc-gtt-ggc-cac-ttg-gag-gag-gag-3′ (SEQ ID NO: 354-357)to generate the Tfi 2M(N)AKK mutant (DNA sequence SEQ ID NO: 358; aminoacid sequence SEQ ID NO: 359). This construct is termed “TfiAKK”.

[0921] To install the “AKK” set of mutations (G502K/E505K/H782A/D783N),into the TscDN 2M (N) construct, (DNA sequence SEQ ID NO: 347) primers959-022-05 to −08:5′-ggg-ctt-ccc-gcc-atc-aag-aag-acg-aag-aag-acg-ggc-aag-cgc-3′,5′-gcg-ctt-gcc-cgt-ctt-ctt-cgt-ctt-ctt-gat-ggc-ggg-aag-ccc-3′,5′-atg-ctt-ttg-cag-gtg-gcc-aac-gaa-ctg-gtc-ctc-3′,5′-gag-gac-cag-ttc-gtt-ggc-cac-ctg-caa-aag-cat-3′ (SEQ ID NO: 360-363)were used to generate Tsc2M(N)AKK (DNA sequence SEQ ID NO: 364; aminoacid sequence SEQ ID NO: 365). This construct is termed “Tsc AKK”.

[0922] Construction of Point Mutants by Recombinant PCR

[0923] Construction of TthAKK(P195A) and TthAKK(P195K)

[0924] To introduce mutations at amino acid position 195 (either a P195Aor P195K) in the nuclease domain of TthAKK construct, mutagenic primer785-073-01 (P195A) 5′-ccc-tcc-gac-aac-ctc-gcc-ggg-gtc-aag-ggc-atc-3′(SEQ ID NO: 370) or 785-073-02 (P195K)5′-ccc-tcc-gac-aac-ctc-aag-ggg-gtc-aag-ggc-atc-3′ (SEQ ID NO: 371) andprimer 209-074-02: 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316)were used in a PCR reaction to generate a 787 base pair fragment.Another PCR fragment was obtained by using the primers: 390-076-085′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 785-073-035′-gag-gtt-gtc-gga-ggg-gtc-3′ (SEQ ID NO: 372) in a reaction with thesame template.

[0925] The two PCR fragments overlap and were combined in a recombinantPCR reaction. The outside primers 390-076-08 and 209-074-02 were added,and the Advantage cDNA PCR kit (Clontech) was used according tomanufacturer's instructions. A 1380 base pair product was generated fromthis reaction.

[0926] The recombinant PCR product was cut with the restriction enzymesEcoRI and NotI to yield a 986 base pair fragment. The TthAKK constructwas prepared by cutting with the same enzymes. The fragment was thenligated into the vector, and transformed into JM109 cells. New mutantsdeveloped from this set of reactions include: Tt AKK(P195A) (DNAsequence SEQ ID NO: 373; amino acid sequence SEQ ID NO: 374) andTthAKK(P195K) (DNA sequence SEQ ID NO: 375; amino acid sequence SEQ IDNO: 376).

[0927] Construction of TthAKK(N417K/L418K)

[0928] The same approach was used to construct TthAKK(N417K/L418K). Twooverlapping PCR fragments were generated by mutagenic primers: 785-73-075′-gag-agg-ctc-cat-cgg-aag-aag-ctt-aag-cgc-ctc-gag-3′ (SEQ ID NO: 377)and 700-10-03 5′-ctt-ctc-tca-tcc-gcc-aaa-aca-gcc-3′ (SEQ ID NO:343), andprimers 158-084-01 5′-ctc-ctc-cac-gag-ttc-ggc-3′ (SEQ ID NO:310) and785-73-08 5′-ccg-atg-gag-cct-ctc-cga-3′ (SEQ ID NO: 378). The twoproducts were combined and amplified with outside primers 700-10-03 and158-084-01. The recombinant PCR product was cut with the restrictionenzymes NotI and BamHI and ligated into the NotI/BamHI pre-cut TthAKKconstruct. This mutant was termed TthAKK(N417K/L418K) (DNA sequence SEQID NO: 379; amino acid sequence SEQ ID NO: 380).

[0929] Construction of Additional TthAKK Point Mutants by Site-DirectedMutagenesis

[0930] Construction of TthAKK(P255L)

[0931] Site specific mutagenesis was performed on the TthAKK constructusing the mutagenic primer 886-049-05 and 886-049-06:5′-gtg-cgc-acc-gac-ctc-ctc-ctg-gag-gtg-gac-ctc-3′ (SEQ ID NO: 381),5′-gag-gtc-cac-ctc-cag-gag-gag-gtc-ggt-gcg-cac-3′ (SEQ ID NO: 382) togenerate TthAKK(P255L) (DNA sequence SEQ ID NO: 383; amino acid sequenceSEQ ID NO: 384).

[0932] Construction of TthAKK(F311Y)

[0933] Site specific mutagenesis was performed on the TthAKK constructusing the mutagenic primer 886-049-09 and 886-049-10:5′-ggg-gcc-ttc-gtg-ggc-tac-gtc-ctc-tcc-cgc-ccc-3′ (SEQ ID NO: P385),5′-ggg-gcg-gga-gag-gac-gta-gcc-cac-gaa-ggc-ccc-3′ (SEQ ID NO: 386) togenerate TthAKK(F311Y) (DNA sequence SEQ ID NO: 387; amino acid sequenceSEQ ID NO: 388).

[0934] Construction of TthAKK(N221H/R224Q)

[0935] Site specific mutagenesis was performed on the TthAKK constructusing the mutagenic primer 886-049-01 and 886-049-02:5′-gaa-aac-ctc-ctc-aag-cac-ctg-gac-cag-gta-aag-cca-gaa-aac-3′ (SEQ IDNO: 389), 5′-gtt-ttc-tgg-ctt-tac-ctg-gtc-cag-gtg-ctt-gag-gag-gtt-ttc-3′(SEQ ID NO: 390) to generate TthAKK(N221H/R224Q) (DNA sequence SEQ IDNO: 391; amino acid sequence SEQ ID NO: 392).

[0936] Construction of TthAKK(R251H)

[0937] Site specific mutagenesis was performed on TthAKK construct usingthe mutagenic primer 886-049-03 and 886-049-04:5′-gag-ctc-tcc-cgg-gtg-cac-acc-gac-ctc-ccc-ctg-3′ (SEQ ID NO: 393),5′-cag-ggg-gag-gtc-ggt-gtg-cac-ccg-gga-gag-ctc-3′ (SEQ ID NO: 394) togenerate TthAKK(R251H) (DNA sequence SEQ ID NO: 395; amino acid sequenceSEQ ID NO: 396).

[0938] Construction of TthAKK(P255L/R251H)

[0939] Site specific mutagenesis was performed on the TthAKK(P255L)construct using the mutagenic primer 886-088-01 and 886-088-02:5′-gag-ctc-tcc-cgg-gtg-cac-acc-gac-ctc-ctc-ctg-3′ (SEQ ID NO: 397),5′-cag-gag-gag-gtc-ggt-gtg-cac-ccg-gga-gag-ctc-3′ (SEQ ID NO: 398) togenerate TthAKK(P255L/R251H) (DNA sequence SEQ ID NO: 399; amino acidsequence SEQ ID NO: 400).

[0940] Construction of Tth AKK L429V (DNA Sequence SEQ ID NO: 401; AminoAcid Sequence SEQ ID NO: 402)

[0941] Palm region randomization mutagenesis was performed on the TthAKK construct using the mutagenic primer 680-79-02 5′-ctc cat cgg aacctc ctt [aag cgc ctc gag ggg gag gag aag ctc ctt tgg] ctc tac cac gaggtg-3′ (SEQ ID NO: 403) and reverse primer 680-79-04 5′-AAG GAG GTT CCGATG GAG-3′ (SEQ ID NO: 404) to introduce the random mutations in thePalm region. Brackets indicate a synthesis of 91% base shown and 3% allother bases.

[0942] Construction of Tth AKK E425V (DNA Sequence SEQ ID NO: 405; AminoAcid Sequence SEQ ID NO: 406)

[0943] Palm region randomization mutagenesis was performed on the TthAKK construct using the mutagenic primer 680-79-02 5′-ctc cat cgg aacctc ctt [aag cgc ctc gag ggg gag gag aag ctc ctt tgg] ctc tac cac gaggtg-3′ (SEQ ID NO: 403) and reverse primer 680-79-04 5′-AAG GAG GTT CCGATG GAG-3′ (SEQ ID NO: 404) to introduce the random mutations in thePalm region. Brackets indicate a synthesis of 91% base shown and 3% allother bases.

[0944] Construction of Tth AKK L422N/E425K (DNA Sequence SEQ ID NO: 407;Amino Acid Sequence SEQ ID NO: 408)

[0945] Palm region randomization mutagenesis was performed on the TthAKK construct using the mutagenic primer 680-79-02 5′-ctc cat cgg aacctc ctt [aag cgc ctc gag ggg gag gag aag ctc ctt tgg] ctc tac cac gaggtg-3′ (SEQ ID NO: 403) and reverse primer 680-79-04 5′-AAG GAG GTT CCGATG GAG-3′ (SEQ ID NO: 404) to introduce the random mutations in thePalm region. Brackets indicate a synthesis of 91% base shown and 3% allother bases.

[0946] Construction of Tth AKK L422F/W430C (DNA Sequence SEQ ID NO: 409;Amino Acid Sequence SEQ ID NO: 410)

[0947] Palm region randomization mutagenesis was performed on Tth AKKDNA using the mutagenic primer 680-79-02 5′-ctc cat cgg aac ctc ctt [aagcgc ctc gag ggg gag gag aag ctc ctt tgg] etc tac cac gag gtg-3′ (SEQ IDNO: 403) and reverse primer 680-79-04 5′-AAG GAG GTT CCG ATG GAG-3′ (SEQID NO: 404) to introduce the random mutations in the Palm region.Brackets indicate a synthesis of 91% base shown and 3% all other bases.

[0948] Construction of Tth AKK A504F (DNA Sequence SEQ ID NO: 411; AminoAcid Sequence SEQ ID NO: 412)

[0949] Site saturation mutagenesis was performed on Tth AKK constructusing the mutagenic primer 680-80-03 5′-cag gag ctt agg ctt ccc nnn ttgaag aag acg aag aag aca-3′ (SEQ ID NO: 413) and reverse primer 680-80-065′-cct aag etc gtc aaa gag-3′ (SEQ ID NO: 414) to introduce the randommutations in the A504 amino acid.

[0950] Construction of Tth AKK A504V (DNA Sequence SEQ ID NO: 415; AminoAcid Sequence SEQ ID NO: 416)

[0951] Site saturation mutagenesis was performed on Tth AKK constructusing the mutagenic primer 680-80-03 5′-cag gag ctt agg ctt ccc nnn ttgaag aag acg aag aag aca-3′ (SEQ ID NO: 413) and reverse primer 680-80-065′-cct aag ctc gtc aaa gag-3′ (SEQ ID NO: 414) to introduce the randommutations in the A504 amino acid.

[0952] Construction of Tth AKK A504S (DNA Sequence SEQ ID NO: 417; AminoAcid Sequence SEQ ID NO: 418)

[0953] Site saturation mutagenesis was performed on Tth AKK constructusing the mutagenic primer 680-80-03 5′-cag gag ctt agg ctt ccc nnn ttgaag aag acg aag aag aca-3′ (SEQ ID NO: 413) and reverse primer 680-80-065′-cct aag ctc gtc aaa gag-3′ (SEQ ID NO: 414) to introduce the randommutations in the A504 amino acid.

[0954] Construction of Tth AKK S517G (DNA Sequence SEQ ID NO: 419; AminoAcid Sequence SEQ ID NO: 420)

[0955] Site saturation mutagenesis was performed on Tth AKK constructusing the mutagenic primer 680-80-07 5′-GGC AAG CGC TCC ACC NNN GCC GCGGTG CTG GAG GCC CTA CGG-3′ (SEQ ID NO: 421) and reverse primer 680-80-105′-GGT GGA GCG CTT GCC-3′ (SEQ ID NO: 422) to introduce the randommutations in the S517 amino acid.

[0956] Construction of Tth AKK A518L (DNA Sequence SEQ ID NO: 423; AminoAcid Sequence SEQ ID NO: 424)

[0957] Site saturation mutagenesis was performed on Tth AKK constructusing the mutagenic primer 680-80-07 5′-GGC AAG CGC TCC ACC AGC NNN GCGGTG CTG GAG GCC CTA CGG-3′ (SEQ ID NO: 425) and reverse primer 680-80-105′-GGT GGA GCG CTT GCC-3′ (SEQ ID NO:422) to introduce the randommutations in the A518 amino acid.

[0958] Construction of Tth AKK A518R (DNA Sequence SEQ ID NO: 426; AminoAcid Sequence SEQ ID NO: 427)

[0959] Site saturation mutagenesis was performed on Tth AKK constructusing the mutagenic primer 680-80-07 5′-GGC AAG CGC TCC ACC AGC NNN GCGGTG CTG GAG GCC CTA CGG-3′ (SEQ ID NO: 425) and reverse primer 680-80-105′-ggt gga gcg ctt gcc-3′ (SEQ ID NO: 422) to introduce the randommutations in the A518 amino acid.

[0960] Construction of Taq5M L451R (DNA Sequence SEQ ID NO: 428; AminoAcid Sequence SEQ ID NO: 429)

[0961] Site directed mutagenesis was performed on the Taq 5M constructusing the mutagenic primer 240-60-055′-acg-ggg-gtg-cgc-cgg-gac-gtg-gcc-tat 3′ (SEQ ID NO: 430) to introducethe L451R mutation in the Taq 5M enzyme.

[0962] Construction of Tth AKK A504K (DNA Sequence SEQ ID NO: 431; AminoAcid Sequence SEQ ID NO: 432)

[0963] Site directed mutagenesis was performed on the Tth AKK constructusing the mutagenic primer 680-69-04 5′-ctt agg ctt ccc aag ttg aag aagacg aag aag aca-3′ (SEQ ID NO: 433) and reverse primer 680-69-05 5′-tgtctt ctt cgt ctt ctt caa ctt ggg aag cct aag-3′ (SEQ ID NO: 434) tointroduce the A504K mutation in the Tth AKK enzyme.

[0964] Construction of Tth AKK H641A (DNA Sequence SEQ ID NO: 435; AminoAcid Sequence SEQ ID NO: 436)

[0965] Site directed mutagenesis was performed on Tth AKK constructusing the mutagenic primer 680-69-08 5′-gag ggg aag gac atc gcc acc cagacc gca agc-3′ (SEQ ID NO: 437) and reverse primer 583-01-02 5′-gct tgcggt ctg ggt ggc gat gtc ctt ccc ctc-3′ (SEQ ID NO: 438) to introduce theH641A mutation in the Tth AKK enzyme.

[0966] Construction of Tth AKK T508P (DNA Sequence SEQ ID NO: 439; AminoAcid Sequence SEQ ID NO: 440)

[0967] Site directed mutagenesis was performed on TthAKK construct usingthe mutagenic primer 680-70-01 5′-ccc-gcc ttg aag aag ccg aag aag acaggc aag-3′ (SEQ ID NO: 441) and reverse primer 680-70-02 5′-ctt gcc tgtctt ctt cgg ctt ctt caa ggc ggg-3′ (SEQ ID NO: 442) to introduce theT508P mutation in the Tth AKK enzyme.

[0968] Chimeras and Mutations in Chimeras.

[0969] Fusion Between TthAKK Enzyme and Alpha-Peptide

[0970] A TthAKK-lacZ-alpha-peptide chimeric fusion was constructed toallow detection of mutations (including frame-shifts, deletions,insertions, etc.) which cause the inability of expression of thefull-length fusion protein based on the colony blue-white screening (Wuet al., Nucleic Acids Research, 24:1710 [1996]).

[0971] Site specific mutagenesis was performed on TthAKK DNA using themutagenic primers 959-041-03,5′-cac-cac-cac-cac-cac-cac-gtc-gac-tag-tgc-tag-cgt-cga-cta-gct-gca-ggc-atg-caa-gt-tgg-c-3′(SEQ ID NO: 477) and 959-041-04,5′-gcc-aag-ctt-gca-tgc-ctg-cag-cta-gtc-gac-gct-agc-act-agt-cga-cgt-ggt-ggt-ggt-ggt-ggt-g-3′(SEQ ID NO: 478) to generate pTthAKK-L with SalI site following the6×His tag at the C-terminus of TthAKK for the insertion of lacZ alphapeptide. The alpha peptide (of 201 amino acids) was first PCR amplifiedfrom the pCRII-TOPO vector (Invitrogen) with primers 959-041-01,5′-cag-gaa-gcg-gcc-gcg-tcg-aca-tga-cca-tga-tta-cgc-caa-gc-3′ (SEQ ID NO:479) and 959-093-01,5′-ggg-ccc-gcc-agg-gtc-gac-tca-ggg-cga-tgg-ccc-act-acg-tga-3′ (SEQ IDNO: 480). The PCR product was then digested with restriction enzyme SalIand ligated into the pTthAKK-L vector, which was also cut with the sameenzyme to generate the chimeric construct TthAKK-alpha peptide (DNAsequence SEQ ID NO: 481; amino acid sequence SEQ ID NO: 482). Theorientation of the insert was confirmed by sequencing.

[0972] Construction of TthTscAKK and TthTfiAKK Enzymes

[0973] The TthAKK construct was cut with the enzymes EcoRI and NotI andthe smaller insert fragment was gel isolated. The TscAKK or TfiAKKconstructs were also cut with EcoRI and NotI and the larger fragment wasgel isolated and purified. The Tth insert (nuclease domain) was ligatedinto the TscAKK and TfiAKK vectors (polymerase domain) to generateTthTscAKK (DNA sequence SEQ ID NO: 447; amino acid sequence SEQ ID NO:448) and TthTfiAKK (DNA sequence SEQ ID NO: 449; amino acid sequence SEQID NO: 450) chimeric constructs.

[0974] FT Mutations of Tth Polymerase to Improve Substrate SpecificityConstruction of Taq(FT)TthAKK

[0975] Site specific mutagenesis was performed on the TaqTthAKKconstruct using the mutagenic primer 473-087-05:5′-cgg-gac-ctc-gag-gcg-cgt-gaa-ccc-cag-gag-gtc-cac-3′ (SEQ ID NO: 483)to introduce the L107F/A108T mutations and generate Taq(FT)TthAKK (DNAsequence SEQ ID NO: 484; amino acid sequence SEQ ID NO: 485).

[0976] Construction of Tfi(FT)TthAKK

[0977] Site specific mutagenesis was performed on the TfiTthAKKconstruct using the mutagenic primers 785-096-01:5′-gtg-gac-ctt-ctg-ggc-ttt-acc-cgc-ctc-gag-gcc-ccg-3′ (SEQ ID NO: 486)and 785-096-02: 5′-cgg-ggc-ctc-gag-gcg-ggt-aaa-gcc-cag-aag-gtc-cac-3′(SEQ ID NO: 487) to introduce the L107F/V108T mutations and generateTfi(FT)TthAKK (DNA sequence SEQ ID NO: 349; amino acid sequence SEQ IDNO: 488).

[0978] Tfi(FT) DN 2M(N) and Tsc(FT) DN 2M(N) Mutants

[0979] The L107F/V108T mutations were introduced by isolating the NotIand SalI fragment of the Tfi DN 2M(N) mutant (DNA sequence SEQ ID NO:345) and inserting it into a NotI-SalI pre-digested Tfi(FT)TthAKK (DNAsequence SEQ ID NO: 349; amino acid sequence SEQ ID NO: 488) to yieldthe Tfi(FT) DN 2M(N) mutant (DNA sequence SEQ ID NO: 350, amino acidsequence SEQ ID NO: 351). To add the L107F or the E108T mutations intothis Tsc-based construct, an identical procedure was done. The NotI andSalI cut fragement of Tsc DN 2M(N) mutant (DNA sequence SEQ ID NO: 348)was inserted into NotI-SalI pre-digested Tsc(FT)TthAKK (DNA sequence SEQID NO: 491) vector to yield Tsc(FT) DN 2M(N) mutant (DNA sequence SEQ IDNO: 352, amino acid sequence SEQ ID NO: 353).

[0980] Construction of Tfi(FT) AKK and Tsc(FT) AKK

[0981] Starting with the Tfi(FT)DN2M(N) construct described previously(DNA sequence SEQ ID NO: 350), primers (959-022-01 to −04:5′-ggc-ctc-acc-ccg-gtg-aag-cgg-acg-aag-aag-acg-ggc-aag-cgc-3′,5′-gcg-ctt-gcc-cgt-ctt-ctt-cgt-ccg-ctt-cac-cgg-ggt-gag-gcc-3′,5′-ctc-ctc-ctc-caa-gtg-gcc-aac-gag-ctg-gtc-ctg-3′,5′-cag-gac-cag-ctc-gtt-ggc-cac-ttg-gag-gag-gag-3′ (SEQ ID NO: 354-357)were used to introduce the “AKK” set of mutations (H784A, G504K andE507K) by site specific mutagenesis. The resulting mutant construct istermed Tfi(FT)AKK (DNA sequence SEQ ID NO:366, amino acid sequence SEQID NO:367).

[0982] Likewise, primers 959-022-05 to −08:5′-ggg-ctt-ccc-gcc-atc-aag-aag-acg-aag-aag-acg-ggc-aag-cgc-3′,5′-gcg-ctt-gcc-cgt-ctt-ctt-cgt-ctt-ctt-gat-ggc-ggg-aag-ccc-3′,5′-atg-ctt-ttg-cag-gtg-gcc-aac-gaa-ctg-gtc-ctc-3′,5′-gag-gac-cag-ttc-gtt-ggc-cac-ctg-caa-aag-cat-3′ (SEQ ID NO: 360-363)were used in a site specific mutagenesis reaction, with theTsc(FT)DN2M(N) mutant (DNA sequence SEQ ID NO: 352) as template togenerate the Tsc(FT)AKK mutant (DNA sequence SEQ ID NO: 368; amino acidsequence SEQ ID NO: 369).

[0983] Construction of Tsc(FT)TthAKK

[0984] Site specific mutagenesis was performed on the TscTthAKKconstruct using the mutagenic primers 785-008-035′-ttt-acc-cgc-ctc-gag-gtg-ccg-ggc-3′ (SEQ ID NO: 489) and reverseprimer 680-21-03 5′-cgg cac ctc gag gcg ggt aaa gcc caa aag gtc cac-3′(SEQ ID NO: 490) to introduce the L107F/E108T mutations and generateTsc(FT)TthAKK (DNA sequence SEQ ID NO: 454; amino acid sequence SEQ IDNO: 491).

[0985] Construction of Taq(FT)TscAKK and Taq(FT)Tf Enzymes

[0986] The Taq(FT)TthAKK construct was cut with the enzymes EcoRI andNotI and the smaller insert fragment was gel isolated. The TscAKK orTfiAKK constructs were also cut with EcoRI and NotI and the largerfragment was gel isolated and purified. The Taq(FT) insert (nucleasedomain) was ligated into the TscAKK and TfiAKK vectors (polymerasedomain) to generate the Taq(FT)TscAKK (DNA sequence SEQ ID NO: 443;amino acid sequence SEQ ID NO: 444) and Taq(FT)TfiAKK (DNA sequence SEQID NO: 445; amino acid sequence SEQ ID NO: 446) chimeric constructs.

[0987] Construction of TaqEFT-Tth(AKK) (DNA Sequence SEQ ID NO:501;Amino Acid Sequence SEQ ID NO: 502)

[0988] Site specific mutagenesis was performed on Taq(FT)-Tth(AKK) DNA(SEQ ID NO: 484) using the mutagenic primers 436-013-08:5′-atc-gtg-gtc-ttt-gac-gcc-gag-gcc-ccc-tcc-ttc-c-3′ (SEQ ID NO:503) and436-013-09 5′-gga-agg-agg-ggg-cct-cgg-cgt-caa-aga-cca-cga-t-3′ (SEQ IDNO:504) to introduce the K70E mutation.

[0989] Additional Mutations to Improve Enzyme Activity of Taq(EFT)TthAKK

[0990] Construction of TaqEFT-Tth(AKK)-A/M1 (DNA Sequence SEQ ID NO:505;Amino Acid Sequence SEQ ID NO: 506)

[0991] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) DNA(SEQ ID NO: 501) using the mutagenic primers 1044-038-01:5′-cag-acc-atg-aat-tcg-gag-gcg-atg-ctg-ccc-ctc-ttt-3′ (SEQ ID NO:507)and 1044-038-₀₂: (SEQ ID NO: 508)5′-aaa-gag-ggg-cag-cat-cgc-ctc-cga-att-cat-ggt-ctg-3′ to introduce theG4EA mutation.

[0992] Construction of TaqEFT-Tth(AKK)-B/M2 (DNA Sequence SEQ ID NO:509;Amino Acid Sequence SEQ ID NO: 510)

[0993] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) DNA(SEQ ID NO: 501) using the mutagenic primers 1044-038-03:5′-gcc-tac-cgc-acc-ttc-ttt-gcc-ctg-aag-ggc-ctc-3 and 1044-038-04:5′-gag-gcc-ctt-cag-ggc-aaa-gaa-ggt-gcg-gta-ggc-3′ (SEQ ID NO: 511 and512, respectively) to introduce the H₂₉F mutation.

[0994] Construction of TaqEFT-Tth(AKK)-CIM3 (DNA Sequence SEQ ID NO:513;Amino Acid Sequence SEQ ID NO: 514)

[0995] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) DNA(SEQ ID NO: 501) using the mutagenic primers 1044-038-05:5′-ctc-ctc-aag-gcc-ctc-aga-gag-gac-ggg-gac-gcg-3′ and 1044-038-06:5′-cgc-gtc-ccc-gtc-ctc-tct-gag-ggc-ctt-gag-gag-3′ (SEQ ID NO: 515 and516, respectively) to introduce the K57R mutation.

[0996] Construction of TaqEFT-Tth(AKK)-D/M5 (DNA Sequence SEQ ID NO:517;Amino Acid Sequence SEQ ID NO:518)

[0997] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) DNA(SEQ ID NO: 501) using the mutagenic primers 1044-038-09:5′-gac-gac-gtc-ctg-gcc-acc-ctg-gcc-aag-aag-gcg-3′ and 1044-038-10:5′-cgc-ctt-ctt-ggc-cag-ggt-ggc-cag-gac-gtc-gtc-3′ (SEQ ID NO: 519 and520, respectively) to introduce the S125T mutation.

[0998] Construction of TaqEFT-Tth(AKK)-E/M6 (DNA Sequence SEQ ID NO:521;Amino Acid Sequence SEQ ID NO: 522)

[0999] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) DNA(SEQ ID NO: 501) using the mutagenic primers 1044-038-11:5′-ggg-gag-aag-acg-gcg-ctc-aag-ctt-ctg-gag-gag-3′ and 1044-038-12:5′-ctc-ctc-cag-aag-ctt-gag-cgc-cgt-ctt-ctc-ccc-3′ (SEQ ID NO: 523 and524, respectively) to introduce the R206L mutation.

[1000] Construction of TaqEFT-Tth(AKK)-F/M7 (DNA Sequence SEQ ID NO:525;Amino Acid Sequence SEQ ID NO: 526)

[1001] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) DNA(SEQ ID NO: 501) using the mutagenic primers 1044-038-13:5′-gag-ccc-gac-cgg-gag-ggg-ctt-aag-gcc-ttt-ctg-gag-agg-3′ and1044-038-14: 5′-cct-ctc-cag-aaa-ggc-ctt-aag-ccc-ctc-ccg-gtc-ggg-ctc-3(SEQ ID NO: 527 and 528, respectively) to introduce the R269G and R271Kmutations.

[1002] Construction of TaqEFT-Tth(AKK)-GIM8 (DNA Sequence SEQ ID NO:529;Amino Acid Sequence SEQ ID NO: 530)

[1003] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) DNA(SEQ ID NO: 501) using the mutagenic primers 1044-038-15:5′-cac-gag-ttc-ggc-ctt-ctg-gga-ggg-gag-aag-ccc-cgg-gag-gag-gcc-ccc-tgg-ccc-3′and 1044-038-16:5′-ggg-cca-ggg-ggc-ctc-ctc-ccg-ggg-ctt-ctc-ccc-tcc-cag-aag-gcc-gaa-ctc-gtg-3′(SEQ ID NO: 531 and 532, respectively) to introduce the E290G, S291G,P292E, A294P, and L295R mutations.

[1004] Construction of TaqEFT-Tth(AKK)-H/M9 (DNA Sequence SEQ ID NO:533;Amino Acid Sequence SEQ ID NO: 534)

[1005] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) DNA(SEQ ID NO: 501) using the mutagenic primers 1044-038-17:5′-ctg-gcc-ctg-gcc-gcc-tgc-agg-ggc-ggc-cgc-gtg-3′ and 1044-038-18:5′-cac-gcg-gcc-gcc-cct-ga-ggc-ggc-cag-ggc-cag-3′ (SEQ ID NO: 535 and536, respectively) to introduce the A328C mutation.

[1006] Construction of TaqEFT-Tth(AKK)-I/M10 (DNA Sequence SEQ IDNO:537; Amino Acid Sequence SEQ ID NO: 538)

[1007] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) DNA(SEQ ID NO: 501) using the mutagenic primers 1080-015-01 (SEQ ID NO:539)5′-ggg gag aag acg gcg agg aag ctt ctg aag gag tgg ggg agc-3′ and1080-015-02 (SEQ ID NO: 540) 5′-gct ccc cca ctc ctt cag aag ctt cct cgccgt ctt ctc ccc-3′ to introduce the E21 OK mutation.

[1008] Construction of TaqEFT-Tth(AKK)-Ml-9 (DNA Sequence SEQ ID NO:541;Amino Acid Sequence SEQ ID NO: 542)

[1009] Seven independent PCR reactions were performed, using constructTaqEFT-Tth(AKK) (SEQ ID: 501) as a template, with the following pairs ofmutagenic primers: PCR Reaction 1; 1044-038-015′-cag-acc-atg-aat-tcg-gag-gcg-atg-ctg-ccc-ctc-ttt-3′ (SEQ ID NO:507)and 1044-038-04 5′-gag-gcc-ctt-cag-ggc-aaa-gaa-ggt-gcg-gta-ggc-3′ (SEQID NO:512) to yield a 108 base pair fragment, PCR Reaction 2;1044-038-03 5′-gcc-tac-cgc-acc-ttc-ttt-gcc-ctg-aag-ggc-ctc-3′ (SEQ IDNO: 511) and 1044-038-065′-cgc-gtc-ccc-gtc-ctc-tct-gag-ggc-ctt-gag-gag-3′ (SEQ ID NO: 516) toyield a 117 base pair fragment, PCR Reaction 3; 1044-038-055′-ctc-ctc-aag-gcc-ctc-aga-gag-gac-ggg-gac-gcg-3′ (SEQ ID NO: 515) and1044-038-10 5′-cgc-ctt-ctt-ggc-cag-ggt-ggc-cag-gac-gtc-gtc-3′ (SEQ IDNO: 520) to yield a 237 base pair fragment, PCR Reaction 4; 1044-038-095′-gac-gac-gtc-ctg-gcc-acc-ctg-gcc-aag-aag-gcg-3′ (SEQ ID NO: 519) and1044-038-12 5′-ctc-ctc-cag-aag-ctt-gag-cgc-cgt-ctt-ctc-ccc-3′ (SEQ IDNO: 524) to yield a 276 base pair fragment, PCR Reaction 5; 1044-038-115′-ggg-gag-aag-acg-gcg-ctc-aag-ctt-ctg-gag-gag-3′ (SEQ ID NO: 523) and1044-038-14 5′-cct-ctc-cag-aaa-ggc-ctt-aag-ccc-ctc-ccg-gtc-ggg-ctc-3′(SEQ ID NO: 528) to yield a 228 base pair fragment, PCR Reaction 6;1044-038-13 5′-gag-ccc-gac-gg-gag-ggg-ctt-aag-gcc-ttt-ctg-gag-agg-3′(SEQ ID NO: 527) and 1044-038-165′-ggg-cca-ggg-ggc-ctc-ctc-ccg-ggg-ctt-ctc-ccc-tcc-cag-aag-gcc-gaa-ctc-gtg-3′(SEQ ID NO: 532) to yield a 113 base pair fragment, PCR Reaction 7;1044-038-155′-cac-gag-ttc-ggc-ctt-ctg-gga-ggg-gag-aag-ccc-cgg-gag-gag-gcc-ccc-tgg-ccc-3′(SEQ ID NO: 531) and 1044-038-185′-cac-gcg-gcc-gcc-cct-gca-ggc-ggc-cag-ggc-cag-3′ (SEQ ID NO: 536) toyield a 157 base pair fragment. The seven PCR products overlap such thatPCR amplification in the absence of primers yielded the appropriate 1005base pair product to introduce the K70E, G4EA, H₂₉F, K57R, S125T, R206L,R269G, R271K, E290G, S291G, P292E, A294P, L295R, and A328C mutations.The product was further amplified using the outside primers: 1044-038-1(SEQ ID NO: 507) and 1044-038-18 (SEQ ID NO: 536) and cloned intopPCR-Script-Amp. From this construct, the nuclease domain was againamplified using primers: 1044-038-1 (SEQ ID NO: 507) and 1044-038-18(SEQ ID NO: 536) and digested with EcoRI and NotI. The digested PCRproduct was ligated into an EcoRI and NotI digested TaqEFT-Tth(AKK)construct and transformed into JM 109 for protein expression andscreening.

[1010] Construction of TaqEFT-Tth(AKK)-M1-10 (DNA Sequence SEQ IDNO:543; Amino Acid Sequence SEQ ID NO: 544)

[1011] Seven independent PCR reactions were performed, using constructTaqEFT-Tth(AKK) (SEQ ID: 501) as a template, with the following pairs ofmutagenic primers: PCR Reaction 1; 1044-038-015′-cag-acc-atg-aat-tcg-gag-gcg-atg-ctg-ccc-ctc-ttt-3′ (SEQ ID NO:507)and 1044-038-04 5′-gag-gcc-ctt-cag-ggc-aaa-gaa-ggt-gcg-gta-ggc-3′ (SEQID NO:512) to yield a 108 base pair fragment, PCR Reaction 2;1044-038-03 5′-gcc-tac-cgc-acc-ttc-ttt-gcc-ctg-aag-ggc-ctc-3′ (SEQ IDNO: 511) and 1044-038-065′-cgc-gtc-ccc-gtc-ctc-tct-gag-ggc-ctt-gag-gag-3′ (SEQ ID NO: 516) toyield a 117 base pair fragment, PCR Reaction 3; 1044-038-055′-ctc-ctc-aag-gcc-ctc-aga-gag-gac-ggg-gac-gcg-3′ (SEQ ID NO: 515) and1044-038-10 5′-cgc-ctt-ctt-ggc-cag-ggt-ggc-cag-gac-gtc-gtc-3′ (SEQ IDNO: 520) to yield a 237 base pair fragment, PCR Reaction 4; 1044-038-095′-gac-gac-gtc-ctg-gcc-acc-ctg-gcc-aag-aag-gcg-3′ (SEQ ID NO: 519) and1080-42-02 5′-gc ttc cag gct ccc cca ctc ctt cag aag ctt gag cgc cgt cttctc ccc-3′ (SEQ ID NO: 546) to yield a 299 base pair fragment, PCRReaction 5; 1080-42-01 5′-ggg gag aag acg gcg ctc agg ctt ctg aag gagtgg ggg agc ctg gaa gc-3′ (SEQ ID NO: 545) and 1044-038-145′-cct-ctc-cag-aaa-ggc-ctt-aag-ccc-ctc-ccg-gtc-ggg-ctc-3′ (SEQ ID NO:528) to yield a 228 base pair fragment, PCR Reaction 6; 1044-038-135′-gag-ccc-gac-cgg-gag-ggg-ctt-aag-gcc-ttt-ctg-gag-agg-3′ (SEQ ID NO:527) and 1044-038-165′-ggg-cca-ggg-ggc-ctc-ctc-ccg-ggg-ctt-ctc-ccc-tcc-cag-aag-gcc-gaa-ctc-gtg-3′(SEQ ID NO: 532) to yield a 113 base pair fragment, PCR Reaction 7;1044-038-155′-cac-gag-ttc-ggc-ctt-ctg-gga-ggg-gag-aag-ccc-cgg-gag-gag-gcc-ccc-tgg-ccc-3′(SEQ ID NO: 531) and 1044-038-185′-cac-gcg-gcc-gcc-cct-gca-ggc-ggc-cag-ggc-cag-3′ (SEQ ID NO: 536) toyield a 157 base pair fragment. The seven PCR products overlap such thatPCR amplification in the absence of primers yielded the appropriate 1005base pair product to introduce the K70E, G4EA, H₂₉F, K57R, S125T, R206L,E210K, R269G, R271K, E290G, S291G, P292E, A294P, L295R, and A328Cmutations. The product was further amplified using the outside primers:1044-038-1 (SEQ ID NO: 507) and 1044-038-18 (SEQ ID NO: 536) and clonedinto pPCR-Script-Amp. From this construct, the nuclease domain was againamplified using primers: 1044-038-1 (SEQ ID NO: 507) and 1044-038-18(SEQ ID NO: 536) and digested with EcoRI and NotI. The digested PCRproduct was ligated into an EcoRI and NotI digested TaqEFT-Tth(AKK)construct and transformed into JM109 for enzyme expression andscreening.

[1012] K69E Mutation of FEN Enzymes to Further Improve SubstrateSpecificity

[1013] Construction of Tth(K69E)AKK, Taq(K69E)TthAKK, Tfi(K69E)TthAKKand Tsc(K69E)TthAKK and Mutants

[1014] Site specific mutagenesis was performed on TthAKK, TaqTthAKK,TfiTthAKK and TscTthAKK DNAs using the mutagenic primers5′-atc-gtg-gtc-ttt-gac-gcc-gag-gcc-ccc-tcc-ttc-c-3′ (SEQ ID NO: 492) and5′-gga-agg-agg-ggg-cct-cgg-cgt-caa-aga-cca-cga-t-3′ (SEQ ID NO: 493) tointroduce the K69E mutation and to generate Tth(K69E)AKK (DNA sequenceSEQ ID NO: 452; amino acid sequence SEQ ID NO: 494), Taq(K69E)ThAKK,(DNA sequence SEQ ID NO: 495; amino acid sequence SEQ ID NO: 496),Tfi(K69E)TthAKK (DNA sequence SEQ ID NO: 497; amino acid sequence SEQ IDNO: 498) and Tsc(K69E)TthAKK (DNA sequence SEQ ID NO: 499; amino acidsequence SEQ ID NO: 500) mutant enzymes.

[1015] Construction of Tsc(167-334)TthAKK

[1016] Two overlapping PCR fragments were generated by primers390-76-08: 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-041-01:5′-ctt-ctc-tca-tcc-gcc-aaa-aca-gcc-3′ (SEQ ID NO: 451) with templateTth(K69E)AKK, (DNA sequence SEQ ID NO: 452), and primers 1044-041-02:5′-ctc-ctc-cac-gag-ttc-ggc-3′ (SEQ ID NO: 453) and 209-074-02:5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) with templateTsc(K69E)TthAKK, (DNA sequence SEQ ID NO: 454). Two products werecombined and amplified with outside primers 390-76-08 and 209-074-02.The recombinant PCR product was cut with the restriction enzymes EcoRIand NotI and ligated into the vector TthAKK which was prepared bycutting with the same enzymes to yield Tsc(167-333)TthAKK construct (DNAsequence SEQ ID NO: 455; amino acid sequence SEQ ID NO: 456).

[1017] Construction of Tsc(222-334)TthAKK

[1018] Two overlapping PCR fragments were generated by primers390-76-08: 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-041-03:5′-ttc-cag-gtg-ctt-gag-gag-gtt-ttc-cag-3′ (SEQ ID NO: 457) with templateTth(K69E)AKK (DNA sequence SEQ ID NO: 452), and primers 1044-041-04:5′-ctc-ctc-aag-cac-ctg-gaa-cag-gtg-aaa-3′ (SEQ ID NO: 458) and209-074-02: 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) withtemplate Tsc(K69E)TthAKK, (DNA sequence SEQ ID NO: 454). Two productswere combined and amplified with outside primers 390-76-08 and209-074-02. The recombinant PCR product was cut with the restrictionenzymes EcoRI and NotI and ligated into the vector TthAKK which wasprepared by cutting with the same enzymes to yield Tsc(222-334)TthAKKconstruct (DNA sequence SEQ ID NO: 459; amino acid sequence SEQ ID NO:460).

[1019] Construction of Tfi(222-334)TthAKK

[1020] Two overlapping PCR fragments were generated by primers390-76-08: 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-058-05:5′-gtc-cag-gtt-ctt-gag-gag-gtt-ttc-cag-3′ (SEQ ID NO: 461) with templateTth(K69E)AKK (DNA sequence SEQ ID NO: 452), and primers 1044-058-06:5′-ctc-ctc-aag-aac-ctg-gac-cgg-gta-aag-3′ (SEQ ID NO: 462) and209-074-02: 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) withtemplate Tfi(K69E)TthAKK (DNA sequence SEQ ID NO: 497). Two productswere combined and amplified with outside primers 390-76-08 and209-074-02. The recombinant PCR product was cut with the restrictionenzymes EcoRI and NotI and ligated into the vector TthAKK which wasprepared by cutting with the same enzymes to yield Tfi(222-334)TthAKKconstruct (DNA sequence SEQ ID NO: 463; amino acid sequence SEQ ID NO:464).

[1021] Construction of Tfi(167-334)TthAKK

[1022] Two overlapping PCR fragments were generated by primers 390-76-085′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-058-075′-gac-gtc-ctt-cgg-ggt-gat-gag-gtg-gcc-3′ (SEQ ID NO: 465) with templateTth(K69E)AKK, (DNA sequence SEQ ID NO: 452), and primers 1044-058-085′-atc-acc-cg-aag-gac-gtc-cag-gag-aag-3′ (SEQ ID NO: 466) and 209-074-025′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) with templateTfi(K69E)TthAKK, (DNA sequence SEQ ID NO: 498). Two products werecombined and amplified with outside primers 390-76-08 and 209-074-02.The recombinant PCR product was cut with the restriction enzymes EcoRIand NotI and ligated into the vector TthAKK which was prepared bycutting with the same enzymes to yield Tfi(167-333)TthAKK construct (DNAsequence SEQ ID NO: 467; amino acid sequence SEQ ID NO: 468).

[1023] Construction of Tsc(111-334)TthAKK

[1024] Two overlapping PCR fragments were generated by primers 390-76-085′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-058-015′-ctc-gag-gcg-ggt-aaa-ccc-cag-gag-gtc-3′ (SEQ ID NO: 469) with templateTth(K69E)AKK (DNA sequence SEQ ID NO: 452), and primers 1044-058-025′-ggg-ttt-acc-cgc-ctc-gag-gtg-ccc-ggc-3′ (SEQ ID NO: 470) and209-074-02 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) withtemplate Tsc(K69E)TthAKK (DNA sequence SEQ ID NO: 499). Two productswere combined and amplified with outside primers 390-76-08 and209-074-02. The recombinant PCR product was cut with the restrictionenzymes EcoRI and NotI and ligated into the vector TthAKK which wasprepared by cutting with the same enzymes to yield Tsc(167-333)TthAKKconstruct (DNA sequence SEQ ID NO: 471; amino acid sequence SEQ ID NO:472).

[1025] Construction of Tsc(1-167)TthAKK

[1026] Two overlapping PCR fragments were generated by primers 390-76-085′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-058-035′-aag-cca-ctc-cgg-ggt-gat-cag-gta-acc-3′ (SEQ ID NO: 473) with templateTsc(K69E)TthAKK (DNA sequence SEQ ID NO: 499), and primers 1044-058-045′-atc-acc-ccg-gag-tgg-ctt-tgg-gag-aag-3′ (SEQ ID NO: 474) and209-074-02 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) withtemplate Tth(K69E)AKK (DNA sequence SEQ ID NO: 452). Two products werecombined and amplified with outside primers 390-76-08 and 209-074-02.The recombinant PCR product was cut with the restriction enzymes EcoRIand NotI and ligated into the vector TthAKK which was prepared bycutting with the same enzymes to yield Tsc(1-167)TthAKK construct (DNAsequence SEQ ID NO: 475; amino acid sequence SEQ ID NO: 476).

[1027] Modification of AfuFEN enzymes

[1028] Construction of pAfuFEN

[1029] Plasmid pAfuFEN1 was prepared as described [U.S. patentapplication Ser. No. 09/684,938, WO 98/23774, each incorporated hereinby reference in their entireties]. Briefly, genomic DNA was preparedfrom one vial (approximately 5 ml of culture) of live A. fulgidusbacteria from DSMZ (DSMZ #4304) with the DNA XTRAX kit (GullLaboratories, Salt Lake City, Utah) according to the manufacturer'sprotocol. The final DNA pellet was resuspended in 100 μl of TE (10 mMTris HCl, pH 8.0, 1 mM EDTA). One microliter of the DNA solution wasemployed in a PCR using the ADVANTAGE cDNA PCR kit (Clonetech); the PCRwas conducted according to manufacturer's recommendations.

[1030] The 5′ end primer is complementary to the 5′ end of the Afu FEN-1gene except it has a 1 base pair substitution to create an Nco I site.The 3′ end primer is complemetary to the 3′ end of the Afu FEN-1 genedownstream from the FEN-1 ORF except it contains a 2 base substitutionto create a Sal I site. The sequences of the 5′ and 3′ end primers are5′-CCGTCAACATTTACCATGGGTGCGGA-3′ (SEQ ID NO:617) and5′-CCGCCACCTCGTAGTCGACATCCTTTTCGTG (SEQ ID NO:618), respectively.Cloning of the resulting fragment was as described for the PfuFEN1 gene,U.S. Pat. No. 5,994,069, incorporated herein in its entirety for allpurposes, to create the plasmid pTrc99-AFFEN1. For expression, thepTrcAfuHis plasmid was constructed by modifying pTrc99-AFFEN1, by addinga histidine tail to facilitate purification. To add this histidine tail,standard PCR primer-directed mutagenesis methods were used to insert thecoding sequence for six histidine residues between the last amino acidcodon of the pTrc99-AFFEN1 coding region and the stop codon. Theresulting plasmid was termed pTrcAfuHis. The protein was then expressedas described and purified by binding to a Ni++ affinity column.

[1031] Construction of Afu(Y236A), A46-5

[1032] Two overlapping PCR fragments were generated from template AfuFEN(SEQ ID NO:556) with primers: 785-73-045′-ctg-gtc-ggg-acg-gac-gcc-aat-gag-ggt-gtg-aag-3′ (SEQ ID NO: 547) and700-10-03 5′-ctt-ctc-tca-tcc-gcc-aaa-aca-gcc-3′ (SEQ ID NO:343), andprimers: 390-076-08 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and785-73-06 5′-gtc-cgt-ccc-gac-cag-aat-3′ (SEQ ID NO: 548). The twoproducts were combined and amplified with outside primers 700-10-03 and390-076-08. The recombinant PCR product was cut with the restrictionenzymes NcoI and SalI and ligated into the AfuFEN construct which wasprepared by cutting with the same enzymes to yield the Afu(Y236A)construct (DNA sequence SEQ ID NO: 549; amino acid sequence SEQ ID NO:550).

[1033] Construction of Afu(Y236R), A56-9

[1034] Two overlapping PCR fragments were generated using the templateAfuFEN with primers: 785-73-055′-ctg-gtc-ggg-acg-gac-agg-aat-gag-ggt-gtg-aag-3′ (SEQ ID NO: 551) and700-10-03 5′-ctt-ctc-tca-tcc-gcc-aaa-aca-gcc-3′ (SEQ ID NO:343), andprimers: 390-076-08 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and785-73-06 5′-gtc-cgt-ccc-gac-cag-aat-3′ (SEQ ID NO: 548). The twoproducts were combined and amplified with outside primers 700-10-03 and390-076-08. The recombinant PCR product was cut with the restrictionenzymes NcoI and SalI and ligated into the AfuFEN construct which wasprepared by cutting with the same enzymes to yield Afu(Y236R) construct(DNA sequence SEQ ID NO: 552; amino acid sequence SEQ ID NO: 553).

[1035] 2. Chimeras of FEN Enzymes and Thermus Polymerase Derivatives

[1036] The following enzyme constructs combine portions of the AfuFENenzyme polymerase domain and the polymerase domain of Thermusploymerases. These combinations were designed based on informationgenerated by molecular modeling.

[1037] Construction of Afu336-Tth296(AKK), AT1-1

[1038] Two overlapping PCR fragments were generated. The first fragmentwas made using the AfuFEN construct (SEQ ID NO:556) as template with theprimers: 390-076-08 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and390-065-05 5′-gaa-cca-cct-ctc-aag-cgt-gg-3′ (SEQ ID NO: 554). The secondfragment was made using TthAKK (SEQ ID NO:558) as template and theprimers: 700-049-015′-acg-ctt-gag-agg-tgg-ttc-ctg-gag-gag-gcc-ccc-tgg-3′ (SEQ ID NO: 555)and 390-076-09 5′-taa-tct-gta-tca-ggc-tg-3′ (SEQ ID NO:557). The twoproducts contain a region of sequence overlap, and were combined andamplified with outside primers 390-076-08 and 390-076-09 in arecombinant PCR reaction. The recombinant PCR product was cut with therestriction enzymes BspEI and SalI and ligated into the AfuFEN constructwhich was prepared by cutting with the same enzymes to yieldAfu336-Tth296(AKK) construct (DNA sequence SEQ ID NO: 559; amino acidsequence SEQ ID NO: 560).

[1039] Construction of Afu328-Tth296(AKK), AT2-3

[1040] Two overlapping PCR fragments were generated, the first byprimers: 390-076-08 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and700-049-02 5′-ggt-tga-ctt-cag-agc-ttt-gag-3′ (SEQ ID NO: 561) withtemplate AfuFEN (DNA sequence SEQ ID NO:556), and the second by primers:700-049-03 5′-aaa-gct-ctg-aag-tca-acc-ctg-gag-gag-gcc-ccc-tgg-3′ (SEQ IDNO: 562) and 390-076-09 5′-taa-tct-gta-tca-ggc-tg-3′ (SEQ ID NO:557)with template TthAKK (DNA sequence SEQ ID NO:558). The two products werecombined and amplified with outside primers 390-076-08 and 390-076-09.The recombinant PCR product was cut with the restriction enzymes BspEIand SalI and ligated into the vector pAfuFEN which was prepared bycutting with the same enzymes to yield Afu336-Tth296(AKK) construct (DNAsequence SEQ ID NO: 563; amino acid sequence SEQ ID NO: 564).

[1041] Construction of Afu336-Taq5M

[1042] The Taq5M construct (SEQ ID NO:41) was cut with the enzymes NotIand SalI and the smaller insert fragment was gel isolated. TheAfu336-Tth296(AKK) construct (DNA sequence SEQ ID NO: 559) was also cutwith the same restriction enzymes and the larger vector fragment waspurified. The insert (Taq5M polymerase domain) was then ligated into thevector to generate the Afu336-Taq5M construct (DNA sequence SEQ ID NO:565; amino acid sequence SEQ ID NO: 566).

[1043] Construction of Afu336-TaqDN

[1044] The TaqDNHT construct was cut with the enzymes NotI and SalI andthe smaller insert fragment was gel isolated. The Afu336-Tth296(AKK)construct (DNA sequence SEQ ID NO: 559) was also cut with the samerestriction enzymes and the larger vector fragment was purified. Theinsert (TaqDN polymerase domain) was then ligated into the vector togenerate the Afu336-Taq5M construct (DNA sequence SEQ ID NO: 567; aminoacid sequence SEQ ID NO: 568).

[1045] Random Chimerization of Thermus Polymerases

[1046] Numerous enzymes with altered functions were generated via randomchimerization of Thermus polymerases based on the principal of DNAshuffling (Volkov and Arnold, Methods in Enzymology 328:456 [2000]). Theprocedure below was used to develop the following chimeras.

[1047] The genes of interest for random chimerization were PCR amplifiedwith primers: 390-076-08 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314)and 209-074-02: 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) togenerate the approximately 1.4 kbp templates. About 2 μg of the DNAtemplates were mixed in equal proportion, and then digested with DNase 1(0.33 U) in a 30 μl reaction at 15° C. for approximately 1 minute togenerate fragments 50-200 bp in size. DNA fragments were purified in a4% agarose gel and extracted by QIAEXII gel extraction kit (QIAGEN)according to manufacturer's instructions. The purified fragments (10 μl)were added to 10 μl of 2×PCR pre-mix (5-fold diluted, cloned Pfu buffer,0.4 mM each dNTP, 0.06 U/μl cloned Pfu DNA polymerase, STRATAGENE Cat.#600153, with accompanying buffer) for the fragment reassembly reaction(PCR program: 3 min 94° C. followed by 40 cycles of 30 sec 94° C., 1 min52° C., 2 min+5 s/cycle 72° C., followed by 4 min at 72° C.). Thereassembled products (1 μl of a 10-fold dilution) were then PCRamplified with a pair of nested primers: 072-090-015′-gag-cgg-ata-aca-att-tca-cac-agg-3′ (SEQ ID NO: 569) and 189-082-015′-tgc-ccg-gtg-cac-gcg-gcc-gcc-cct-gca-ggc-3′ (SEQ ID NO: 570) using theCLONTECH GC melt cDNA PCR kit (Cat.# K1907-Y) according to themanufacturer's instructions. The purified PCR products were digestedwith restriction enzymes EcoRI and NotI and then ligated into the TthAKKconstruct that was prepared by cutting with the same enzymes. Theligation mixture was transformed into JM109 competent cells and colonieswere screened for enzyme activity.

[1048] 1. Generation of Random Chimeras S26 and S36

[1049] The templates used to develop these chimeric enzymes were thenuclease domains from TthAKK, TaqTthAKK, TscTthAKK and the TfiTthAKKconstructs. Two clones were found to show improvement of activity basedon primary activity screening. Random chimeras S26 (DNA sequence SEQ IDNO: 571; amino acid sequence SEQ ID NO: 572) and S36 (DNA sequence SEQID NO: 573; amino acid sequence SEQ ID NO: 574) were then sequenced andisolated.

[1050] 2. Introduction of L107F/E108T, L109FV101T and K69E Mutations inS26 and S36 to Improve Substrate Specificity

[1051] Construction of S26(FT)

[1052] Site specific mutagenesis was performed on pS26 DNA using themutagenic primers: 785-008-03 5′-ttt-acc-cgc-ctc-gag-gtg-cg-ggc-3′ (SEQID NO: 489) and 680-21-035′-cgg-cac-ctc-gag-gcg-ggt-aaa-gcc-caa-aag-gtc-cac-3′ (SEQ ID NO: 490)to introduce the L107F/E108T mutations and generate S26(FT) (DNAsequence SEQ ID NO: 575; amino acid sequence SEQ ID NO: 576).

[1053] Construction of S36(FT)

[1054] Site specific mutagenesis was performed on pS36 DNA using themutagenic primers: 785-096-01:5′-gtg-gac-ctt-ctg-ggc-ttt-acc-cgc-ctc-gag-gcc-ccg-3′ (SEQ ID NO: 486)and 785-096-02: 5′-cgg-ggc-ctc-gag-gcg-ggt-aaa-gcc-cag-aag-gtc-cac-3′(SEQ ID NO: 487) to introduce the L109FV110T mutations and generateS36(FT) (DNA sequence SEQ ID NO: 577; amino acid sequence SEQ ID NO:578).

[1055] Construction of S26(K69E)

[1056] Site specific mutagenesis was performed on S26 DNA using themutagenic primers: 5′-atc-gtg-gtc-ttt-gac-gcc-gag-gcc-ccc-tcc-ttc-c-3′(SEQ ID NO: 492) and 5′-gga-agg-agg-ggg-cct-cgg-cgt-caa-aga-cca-cga-t-3′(SEQ ID NO: 493) to introduce the K69E mutation and to generateS26(K69E) (DNA sequence SEQ ID NO: 579; amino acid sequence SEQ ID NO:580).

[1057] Construction of S26(FT/K69E)

[1058] Site specific mutagenesis was performed on S26(FT) DNAs using themutagenic primers: 5′-atc-gtg-gtc-ttt-gac-gcc-gag-gcc-ccc-tcc-ttc-c-3′(SEQ ID NO: 492) and 5′-gga-agg-agg-ggg-cct-cgg-cgt-caa-aga-cca-cga-t-3′(SEQ ID NO: 493) to introduce the K69E mutation and to generateS26(FT/K69E) (DNA sequence SEQ ID NO: 581; amino acid sequence SEQ IDNO: 582).

[1059] 3. More Random Chimeras, N3D7, N1A12 N1C4, and N2C3

[1060] The templates used to generate these chimerics were the nucleasedomains of Tth(K69E)AKK, Taq(K69E)TthAKK, Tsc(K69E)TthAKK andTfi(K69E)TthAKK constructs. Four clones were shown to have improvedactivity based on the primary activity screening. Random chimeras N3D7(DNA sequence SEQ ID NO: 583; amino acid sequence SEQ ID NO: 584), NlA12(DNA sequence SEQ ID NO: 585; amino acid sequence SEQ ID NO: 586), N1C4(DNA sequence SEQ ID NO: 587; amino acid sequence SEQ ID NO: 588) andN2C3 (DNA sequence SEQ ID NO: 589; amino acid sequence SEQ ID NO:590)were then sequenced and isolated respectively.

[1061] Generation of G, L and GL Variants

[1062] A variant of Tth polymerase having an Asp to Gly change at aminoacid 385 (D385G) and an Arg toLeu change at amino acid 455 (R455L),(amino acid SEQ ID NO:2712; gene sequence SEQ ID NO:2713) was used tocreate derivatives and chimerical enzymes. Constructs comprising thesetwo amino acid variations are indicated by the ‘GL’ designation, below.Chimeras comprising only one of the variations are indicated as either‘G’ or ‘L’. To create a non-polymerizing variant, the sequence encodingan aspartic acid at position 787 was changed by site-specificmutagenesis as described above to a sequence encoding asparagine.Mutagenesis of pTrcTth GL-2 with the following oligonucleotide:5′-CAGGAGGAGCTCGTTGTGGACCTGGA-3′ (SEQ ID NO:260) was performed to createthe plasmid pTrcTthDN GL. The mutant protein and coding nucleic acidsequence is termed TthDN GL, SEQ ID NOS:2714 and 2715 respectively.

[1063] A. Tth DN HT GL

[1064] Six amino acid histidine tags (his-tags) were added onto thecarboxy termini of Tth DN GL, as described above for Tth DN. Theresulting mutant gene is termed Tth DN HT GL (SEQ ID NO:2716, nucleicacid sequence; SEQ ID NO:2717, amino acid sequence). The protein wasexpressed and purified as described for Tth DN HT, above.

[1065] B. Generation of Tth DN RX HT GL

[1066] Mutagenesis was performed to introduce 3 additional, uniquerestriction sites into the polymerase domain of Tth DN HT enzymes.Site-specific mutagenesis was performed using the TransformerSite-Directed Mutagenesis Kit from (Clonetech) according tomanufacturer's instructions. One of two different selection primers,Trans Oligo AlwNI/SpeI or Switch Oligo SpeI/AlwNI (Clontech, Palo AltoCalif. catalog #6488-1 or catalog #6373-1) was used for all mutagenesisreactions described. The selection oligo used in a given reaction isdependent on the selection restriction site present in the vector. Allmutagenic primers were synthesized by standard synthetic chemistry.Resultant colonies were expressed in E.coli strain JM109.

[1067] The Not I site (amino acid position 328) was created using themutagenic primers 5′-gccgccaggggcggccgcgtccaccgggcc (SEQ ID NO:269) and5′-gcctgcaggggcggccgcgtgcaccggggca (SEQ ID NO:270) corresponding to thesense strand of the Tth DN HT GL gene. The BstI (amino acid position382) and NdeI (amino acid position 443) sites were introduced into thegene using sense strand mutagenic primes 5′-ctcctggacccttcgaacaccacccc(SEQ ID NO:271) and 5′-gtcctggcccatatggaggccac (SEQ ID NO:272). Themutant plasmid was over-expressed and purified using Qiagen QiaPrep SpinMini Prep Kit (cat. # 27106). The vector were tested for the presence ofthe restriction sites by DNA sequencing and restriction mapping. Thisconstruct is termed Tth DN RX HT GL (DNA sequence SEQ ID NO:2718; aminoacid SEQ ID NO:2719). Chimeric constructs were created using Tth DN RXHT GL.

[1068] 1. Construction of TaqTth(N) GL

[1069] The first exchange that was performed involved the polymerasedomains of the two enzymes. Separation of the nuclease domain (theN-terminal end of the protein) from the polymerase domain (theC-terminal portion of the protein) was accomplished by cutting bothgenes with the restriction endonucleases EcoRI and NotI. Theapproximately 900 base pair fragment from the Taq DN RX HT gene wascloned into the homologous sites of the Tth DN RX HT GL gene, yieldingthe chimera TaqTth(N) GL (DNA sequence SEQ ID NO:2675; amino acidsequence SEQ ID NO:2641) which has the Taq DN RX HT 5′ nuclease domainand the Tth DN RX HT GL polymerase domain.

[1070] 2. Construction of TaqTth(N-B) GL

[1071] The Taq DN RX HT construct was cut with the enzymes NdeI andBamHI and the larger, vector fragment was gel isolated as detailedabove. The Tth DN RX HT GL construct was also cut with NdeI and BamHIand the smaller (approximately 795 base pairs) Tth fragment was gelisolated and purified. The Tth GL NdeI-BamHI insert was ligated into theTaq NdeI-BamHI vector as detailed above to generate the TaqTth(N-B) GL(DNA sequence SEQ ID NO:2676; amino acid sequence SEQ ID NO:2642).

[1072] 3. Construction of TaqTth(N-D) G

[1073] The Taq DN RX HT construct was cut with the enzymes NotI and NdeIand the larger vector fragment was isolated as detailed above. The TthDN RX HT GL construct was also cut with NotI and NdeI and the smaller(approximately 345 base pairs) Tth fragment was gel isolated andpurified. The Tth GL NotI-NdeI insert was ligated into the Taq NotI-NdeIvector as detailed above to generate the TaqTth(N-D) G (DNA sequence SEQID NO:2677; amino acid sequence SEQ ID NO:2643).

[1074] 4. Construction of TaqTth(D-B) L

[1075] The Taq DN RX HT construct was cut with the enzymes NdeI andBamHI and the larger vector fragment was isolated as detailed above. TheTth DN RX HT GL construct was also cut with NdeI and BamHI and thesmaller (approximately 450 base pairs) Tth fragment was gel isolated andpurified. The Tth GL NdeI-BamHI insert was ligated into the TaqNdeI-BamHI vector as detailed above to generate the TaqTth(D-B) L (DNAsequence SEQ ID NO:2678; amino acid sequence SEQ ID NO:2644).

[1076] 5. Construction of TaqTth(Bs-B) L

[1077] The Taq DN RX HT construct was cut with the enzymes BstBI andBamHI and the larger vector fragment was isolated as detailed above. TheTth DN RX HT GL construct was also cut with BstBI and BamHI and thesmaller (approximately 633 base pairs) Tth fragment was gel isolated andpurified. The Tth GL NdeI-BamHI insert was ligated into the TaqNdeI-BamHI vector as detailed above to generate TaqTth(Bs-B) L (DNAsequence SEQ ID NO:2679; amino acid sequence SEQ ID NO:2645).

[1078] 6. Construction of TaqTth(N-Bs) G

[1079] The Taq DN RX HT construct was cut with the enzymes NotI andBstBI and the larger vector fragment was isolated as detailed above. TheTth DN RX HT GL construct was also cut with NotI and BstBI and thesmaller (approximately 162 base pairs) Tth fragment was gel isolated andpurified. The Tth GL NotI-BstBI insert was ligated into the TaqNotI-BstBI vector as detailed above to generate TaqTth(N-Bs) G (DNAsequence SEQ ID NO:2680; amino acid sequence SEQ ID NO:2646).

[1080] 7. Construction of TthTaq(B-S) GL

[1081] The Tth DN RX HT GL construct was cut with the enzymes BamHI andSalI and the larger vector fragment was isolated as detailed above. TheTaq DN RX HT construct was also cut with BamHI and SalI and the smaller(approximately 741 base pairs) Taq fragment was gel isolated andpurified. The Taq BamHI-SalI insert was ligated into the Tth GLBamHI-SalI vector as detailed above to generate the TthTaq(B-S) GL (DNAsequence SEQ ID NO:2681; amino acid sequence SEQ ID NO:2647).

[1082] 8. Construction of TaqTth(D-B) L E404H (DNA Sequence SEQ IDNO:2684; Amino Acid Sequence SEQ ID NO:2650)

[1083] Site specific mutagenesis was performed on pTrc99A TaqTth(D-B) LDNA using the mutagenic primer 240-60-01 5′-gag gag gcg ggg cac cgg gccgcc ctt-3′ (SEQ ID NO:277) to introduce the E404H mutation.

[1084] 9. Construction of TaqTth(D-B) L F413H/A414R (DNA Sequence SEQ IDNO:2685; Amino Acid Sequence SEQ ID NO:2651)

[1085] Site specific mutagenesis was performed on pTrc99A TaqTth(D-B) LDNA using the mutagenic primer 240-60-02 5′-ctt tcc gag agg ctc cat cggaac ctg tgg ggg agg-3′ (SEQ ID NO:278) to introduce the F413H and theA414R mutations.

[1086] 10. Construction of TaqTth(D-B) L W417L/G418K (DNA Sequence SEQID NO:2686; Amino Acid Sequence SEQ ID NO:2652)

[1087] Site specific mutagenesis was performed on pTrc99A TaqTth(D-B) LDNA using the mutagenic primer 240-60-03 5′-ctc ttc gcc aac ctg ctt aagagg ctt gag ggg gag-3′ (SEQ ID NO:279) to introduce the W417L and theG418K mutations.

[1088] 11. Construction of TaqTth(D-B) L A439R (DNA Sequence SEQ IDNO:2687; Amino Acid Sequence SEQ ID NO:2653)

[1089] Site specific mutagenesis was performed on pTrc99A TaqTth(D-B)DNA using the mutagenic primer 240-60-04 5′-agg ccc ctt tcc egg gtc ctggcc cat-3′ (SEQ ID NO:280) to introduce the A439R mutation.

[1090] 12. Construction of TaqTth(N-D) G L451R (DNA Sequence SEQ IDNO:2688; Amino Acid Sequence SEQ ID NO:2654)

[1091] Site specific mutagenesis was performed on pTrc99A TaqTth(N-D) GDNA using the mutagenic primer 240-60-05 5′-acg ggg gtg cgc egg gac gtggec tat-3′ (SEQ ID NO:281) to introduce the L415 mutation.

[1092] 13. Construction of TaqTth(N-D) G R457Q (DNA Sequence SEQ IDNO:2689; Amino Acid Sequence SEQ ID NO:2655)

[1093] Site specific mutagenesis was performed on pTrc99A TaqTth(N-D) GDNA using the mutagenic primer 240-60-06 5′-gtg gcc tat ctc cag gcc ttgtcc ctg-3′ (SEQ ID NO:282) to introduce the L415Q mutation.

[1094] 14. Construction of TaqTth(N-D) G V463L (DNA Sequence SEQ IDNO:2690; Amino Acid Sequence SEQ ID NO:2656)

[1095] Site specific mutagenesis was performed on pTrc99A TaqTth(N-D) GDNA using the mutagenic primer 240-60-07 5′-ttg tcc ctg gag ctt gcc gaggag atc-3′ (SEQ ID NO:283) to introduce the V463L mutation.

[1096] 15. Construction of TaqTth(N-D) G A468R (DNA Sequence SEQ IDNO:2691; Amino Acid Sequence SEQ ID NO:2657)

[1097] Site specific mutagenesis was performed on pTrc99A TaqTth(N-D) GDNA using the mutagenic primer 240-60-08 5′-gcc gag gag atc cgc cgc ctcgag gcc-3′ (SEQ ID NO:284) to introduce the A468R mutation.

[1098] 16. Construction of TaqTth(N-D) G A472E (DNA Sequence SEQ IDNO:2692; Amino Acid Sequence SEQ ID NO:2658)

[1099] Site specific mutagenesis was performed on pTrc99A TaqTth(N-D) GDNA using the mutagenic primer 240-60-09 5′-gcc cgc etc gag gag gag gtette cgc-3′ (SEQ ID NO:285) to introduce the A472E mutation.

[1100] 17. Construction of TaqTth(N-D) G G499R (DNA Sequence SEQ IDNO:2693; Amino Acid Sequence SEQ ID NO:2659)

[1101] Site specific mutagenesis was performed on pTrc99A TaqTth(N-D) GDNA using the mutagenie primer 240-60-10 5′-ttt gac gag cta agg ctt cccgcc atc-3′ (SEQ ID NO:286) to introduce the G499R mutation.

[1102] 18. Construction of TaqTth(N-D) G E507Q (DNA Sequence SEQ IDNO:2694; Amino Acid Sequence SEQ ID NO:2660)

[1103] Site specific mutagenesis was performed on pTre99A TaqTth(N-D) GDNA using the mutagenie primer 276-046-04 5′-atc gec aag acg caa aag accggc aag-3′ (SEQ ID NO:287) to introduce the E507Q mutation.

[1104] 19. Construction of TaqTth(N-D) G Y535H (DNA Sequence SEQ IDNO:2695; Amino Acid Sequence SEQ ID NO:2661)

[1105] Site specific mutagenesis was performed on pTre99A TaqTth(N-D) GDNA using the mutagenie primer 240-60-11 5′-aag ate ctg cag cac cgg gagctc acc-3′ (SEQ ID NO:288) to introduce the Y535H mutation.

[1106] 20. Construction of TaqTth(N-D) G S543N (DNA Sequence SEQ IDNO:2696; Amino Acid Sequence SEQ ID NO:2662)

[1107] Site specific mutagenesis was performed on pTrc99A TaqTth(N-D) GDNA using the mutagenic primer 240-60-12 5′-acc aag ctg aag aac acc tacatt gac-3′ (SEQ ID NO:289) to introduce the S543N mutation.

[1108] 21. Construction of TaqTth(N-D) G 1546V (DNA Sequence SEQ IDNO:2697; Amino Acid Sequence SEQ ID NO:2663)

[1109] Site specific mutagenesis was performed on pTrc99A TaqTth(N-D) GDNA using the mutagenic primer 240-60-13 5′-aag agc acc tac gtg gac cccttg ccg-3′ (SEQ ID NO:290) to introduce the 1546V mutation.

[1110] 22. Construction of TaqTth(N-D) G D551S/I553V (DNA Sequence SEQID NO:2698; Amino Acid Sequence SEQ ID NO:2664)

[1111] Site specific mutagenesis was performed on pTrc99A TaqTth(N-D) GDNA using the mutagenic primer 240-60-14 5′-att gac ccc ttg ccg agc ctcgtc cac ccc agg acg ggc-3′ (SEQ ID NO:291) to introduce the D551S andthe 1553V mutations.

[1112] 23. Construction of Tth DN RX HT GL H641A, Tth DN RX HT GL H748A,Tth DN RX HT GL H786A

[1113] Site specific mutagenesis was performed on pTrc99A Tth DN RX HTGL DNA using the mutagenic primer 583-001-02: 5′-gct tgc ggt ctg ggt ggcgat gtc ctt ccc ctc-3′ (SEQ ID NO:294) to introduce the H641A mutation(DNA sequence SEQ ID NO:2699; amino acid sequence SEQ ID NO:2665), orthe mutagenic primer 583-001-03: 5′ cat gtt gaa ggc cat ggc ctc cgc ggcctc cct-3′ (SEQ ID NO:295) to generate the H748A mutant (DNA sequenceSEQ ID NO:2700; amino acid sequence SEQ ID NO:2666), or the mutagenicprimer 583-001-04: 5′-cag gag gag ctc gtt ggc gac ctg gag gag-3′ (SEQ IDNO:296) to generate the H786A mutant enzyme (DNA sequence SEQ IDNO:2701; amino acid sequence SEQ ID NO:2667).

[1114] 24. Construction of Tth DN RX HT GL (H786A/G506K/Q509K)

[1115] Starting with the mutant Tth DN RX HT GL H786A, generated above,site specific mutagenesis was done using the mutagenic primer604-022-02: 5′-gga gcg ctt gcc tgt ctt ctt cgt ctt ctt caa ggc ggg aggcct-3′ (SEQ ID NO:297) to generate this variant termed “TthAKK GL”, (DNAsequence SEQ ID NO:2702; amino acid sequence SEQ ID NO:2668).

[1116] 25. TthDN RX HT H786A Random Mutagenesis

[1117] To generate mutants in the helix-hairpin-helix region of theTthDN RX HT H786A GL (SEQ ID NO:2667) enzyme, two different PCRreactions were performed using the H786A (SEQ ID NO:2701) mutant as atemplate. Starting with TthDN GL H786A discussed above, and using primer604-08-06: 5′-gtc gga ggg gtc ccc cac gag-3′ (SEQ ID NO:313) and primer390-76-08: 5′-tgt gga att gtg agc gg (SEQ ID NO:314), a 620 base pairPCR fragment was generated. PCR reactions were performed using theAdvantage cDNA PCR kit (Clontech) according to manufacturer'sinstructions. This PCR product includes amino acids 1-194. No mutationswere introduced via this reaction, however the restriction enzyme siteEcoRI is present at the 5′ end.

[1118] Starting with TthDN RX HT GL H786A discussed above, and usingmutagenic primer 604-08-05: 5′-ctc gtg ggg gac ccc tcc gac aac ctc (cccggg gtc aag ggc atc ggg gag aag acc gcc) ctc aag ctt ctc aag-3′ (SEQ IDNO:315) and primer 209-74-02: 5′-gtg gcc tcc ata tgg gcc agg α-3′ (SEQID NO:316) a 787 base pair PCR fragment was generated. PCR reactionswere done as above. This fragment does contain random mutations, due tothe presence of the mutagenic primer, 604-08-05. The bases within theparenthesis of this primer were synthesized such that 91% of thesequence is wild-type, while the additional 9% is evenly divided betweenthe remaining 3 bases.

[1119] The two PCR fragments overlap, and were combined in a recombinantPCR reaction. Primers 390-76-08 and 209-74-02 were added, and theAdvantage cDNA PCR kit (Clontech) was again used according tomanufacturer's instructions. A 1380 base pair product was generated fromthis reaction.

[1120] The recombinant PCR product was cut with the restriction enzymesEcoRI and NotI according to the manufacturer's instructions to yield a986 base pair fragment. TthDN RX HT GL H786A was prepared by cuttingwith the same enzymes. The fragment was then ligated into the vector,and transformed into JM109 cells. New mutants developed from this set ofreactions include:

[1121] TthDN RX HT GL H786A/P197R/K200R (DNA sequence SEQ ID NO:2703;amino acid sequence SEQ ID NO:2669).

[1122] TthDN RX HT GL H786A/K205Y (DNA sequence SEQ ID NO:2704; aminoacid sequence SEQ ID NO:2670).

[1123] TthDN RX HT GL H786A/G203R (DNA sequence SEQ ID NO:2705; aminoacid sequence SEQ ID NO:2671).

[1124] 26. Construction of TaqTthAKK GL (DNA Sequence SEQ ID NO:2706;Amino Acid Sequence SEQ ID NO:2672)

[1125] Chimeric mutant TaqTthAKK GL was generated by cutting Tth DN RXHT GL (H786A/G506K/Q509K) (SEQ ID NO:2702; here abbreviated TthAKK GL)and Taq 4M G504 (SEQ ID NO:108; here abbreviated Taq 5M) with therestriction endonucleases EcoRI and NotI. The smaller insert fragment ofTaq 5M and the larger vector fragment of Tth AKK GL were gel purified asdetailed in Example 3D, and the insert fragments were ligated asdescribed in Example 3D. Screening and verification of the constructsequence was also done as in Example 3D.

[1126] 27. Construction of TfiTth AKK GL (DNA Sequence SEQ ID NO:2707;Amino Acid Sequence SEQ ID NO:2673), TscTthAKK GL (DNA Sequence SEQ IDNO:2708; Amino Acid Sequence SEQ ID NO:2674)

[1127] To generate chimeric enzymes between TthAKK GL (SEQ ID NO:2702),and Tfi DN 2M (SEQ ID NO:130), or Tsc DN 2M (SEQ ID NO:131), Tth DN RXHT GL (H786A/G506K/Q509K) DNA (SEQ ID NO:2702) was cut with EcoRI andNotI, and the larger vector fragment was gel isolated as above. The 993base pair Eco RI-NotI fragements of Tfi and Tsc were prepared asdescribed in Example 8. Ligations were performed as detailed in Example3D, as was the screening and verification of the new constructs.

[1128] 28. Construction of TthAKK GL (P195A) and TthAKK GL (P195K)

[1129] To introduce mutations at amino acid position 195 (either a P195Aor P195K) in the nuclease domain of TthAKK GLconstruct, mutagenic primer785-073-01 (P195A) 5′-ccc-tcc-gac-aac-ctc-gcc-ggg-gtc-aag-ggc-atc-3′(SEQ ID NO: 370) or 785-073-02 (P195K)5′-ccc-tcc-gac-aac-tc-aag-ggg-gtc-aag-ggc-atc-3′ (SEQ ID NO: 371) andprimer 209-074-02: 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316)were used in a PCR reaction to generate a 787 base pair fragment.Another PCR fragment was obtained by using the primers: 390-076-085′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 785-073-035′-gag-gtt-gtc-gga-ggg-gtc-3′ (SEQ ID NO: 372) in a reaction with thesame template.

[1130] The two PCR fragments overlap and were combined in a recombinantPCR reaction. The outside primers 390-076-08 and 209-074-02 were added,and the Advantage cDNA PCR kit (Clontech) was used according tomanufacturer's instructions. A 1380 base pair product was generated fromthis reaction.

[1131] The recombinant PCR product was cut with the restriction enzymesEcoRI and NotI to yield a 986 base pair fragment. The TthAKK GLconstructwas prepared by cutting with the same enzymes. The fragment was thenligated into the vector, and transformed into JM109 cells. New mutantsdeveloped from this set of reactions include: TthAKK GL(P195A) (DNAsequence SEQ ID NO: 2721; amino acid sequence SEQ ID NO: 2722) andTthAKK GL (P195K) (DNA sequence SEQ ID NO: 723; amino acid sequence SEQID NO:2724).

[1132] 29. Construction of TthAKK GL (N417K/L418K)

[1133] The same approach was used to construct TtbAKK GL (N417K/L418K).Two overlapping PCR fragments were generated by mutagenic primers:785-73-07 5′-gag-agg-ctc-cat-cgg-aag-aag-ctt-aag-cgc-ctc-gag-3′ (SEQ IDNO: 377) and 700-10-03 5′-ctt-ctc-tca-tcc-gcc-aaa-aca-gcc-3′ (SEQ IDNO:343), and primers 158-084-01 5′-ctc-ctc-cac-gag-ttc-ggc-3′ (SEQ IDNO:310) and 785-73-08 5′-ccg-atg-gag-cct-ctc-cga-3′ (SEQ ID NO: 378).The two products were combined and amplified with outside primers700-10-03 and 158-084-01. The recombinant PCR product was cut with therestriction enzymes NotI and BamHI and ligated into the NotI/BamHIpre-cut TthAKK GL construct. This mutant was termed TthAKK GL(N417K/L418K) (DNA sequence SEQ ID NO: 2725; amino acid sequence SEQ IDNO: 2726).

[1134] 30. Construction of TthAKK GL (P255L)

[1135] Site specific mutagenesis was performed on the TthAKK GLconstruct using the mutagenic primer 886-049-05 and 886-049-06:5′-gtg-cgc-acc-gac-ctc-ctc-ctg-gag-gtg-gac-ctc-3′ (SEQ ID NO: 381),5′-gag-gtc-cac-ctc-cag-gag-gag-gtc-ggt-gcg-cac-3′ (SEQ ID NO: 382) togenerate TthAKK GL (P255L) (DNA sequence SEQ ID NO: 2727; amino acidsequence SEQ ID NO: 2728).

[1136] 31. Construction of TthAKK GL (F311Y)

[1137] Site specific mutagenesis was performed on the TthAKK GLconstruct using the mutagenic primer 886-049-09 and 886-049-10:5′-ggg-gcc-ttc-gtg-ggc-tac-gtc-ctc-tcc-cgc-ccc-3′ (SEQ ID NO: P385),5′-ggg-gcg-gga-gag-gac-gta-gcc-cac-gaa-ggc-ccc-3′ (SEQ ID NO: 386) togenerate TthAKK GL (F311Y) (DNA sequence SEQ ID NO:2729; amino acidsequence SEQ ID NO:2730).

[1138] 32. Construction of TthAKK GL (N221H/R224Q)

[1139] Site specific mutagenesis was performed on the TthAKK GLconstruct using the mutagenic primer 886-049-01 and 886-049-02:5′-gaa-aac-ctc-ctc-aag-cac-ctg-gac-cag-gta-aag-cca-gaa-aac-3′ (SEQ IDNO: 389), 5′-gtt-ttc-tgg-ctt-tac-ctg-gtc-cag-gtg-ctt-gag-gag-gtt-ttc-3′(SEQ ID NO: 390) to generate TthAKK GL (N221H/R224Q) (DNA sequence SEQID NO: 2731; amino acid sequence SEQ ID NO: 2732).

[1140] 33. Construction of TthAKK GL (R251H)

[1141] Site specific mutagenesis was performed on TthAKK GL constructusing the mutagenic primer 886-049-03 and 886-049-04:5′-gag-ctc-tcc-cgg-gtg-cac-acc-gac-ctc-ccc-ctg-3′ (SEQ ID NO: 393),5′-cag-ggg-gag-gtc-ggt-gtg-cac-ccg-gga-gag-ctc-3′ (SEQ ID NO: 394) togenerate TthAKK GL (R251H) (DNA sequence SEQ ID NO:2733; amino acidsequence SEQ ID NO:2734).

[1142] 34. Construction of TthAKK GL (P255L/R251H)

[1143] Site specific mutagenesis was performed on the TthAKK GL (P255L)construct using the mutagenic primer 886-088-01 and 886-088-02:5′-gag-ctc-tcc-cgg-gtg-cac-acc-gac-ctc-ctc-ctg-3′ (SEQ ID NO: 397),5′-cag-gag-gag-gtc-ggt-gtg-cac-ccg-gga-gag-ctc-3′ (SEQ ID NO: 398) togenerate TthAKK GL (P255L/R251H) (DNA sequence SEQ ID NO:2735; aminoacid sequence SEQ ID NO:2736).

[1144] 35. Construction of Tth AKK GL L429V (DNA Sequence SEQ IDNO:2737; Amino Acid Sequence SEQ ID NO:2738)

[1145] Palm region randomization mutagenesis was performed on the TthAKK GL construct using the mutagenic primer 680-79-02 5′-ctc cat cgg aacctc ctt [aag cgc ctc gag ggg gag gag aag ctc ctt tgg] ctc tac cac gaggtg-3′ (SEQ ID NO: 403) and reverse primer 680-79-04 5′-AAG GAG GTT CCGATG GAG-3′ (SEQ ID NO: 404) to introduce the random mutations in thePalm region. Brackets indicate a synthesis of 91% base shown and 3% allother bases.

[1146] 36. Construction of Tth AKK GL E425V (DNA Sequence SEQ IDNO:2739; Amino Acid Sequence SEQ ID NO:2740)

[1147] Palm region randomization mutagenesis was performed on the TthAKK GL construct using the mutagenic primer 680-79-02 5′-ctc cat cgg aacctc ctt [aag cgc ctc gag ggg gag gag aag ctc ctt tgg] ctc tac cac gaggtg-3′ (SEQ ID NO: 403) and reverse primer 680-79-04 5′-AAG GAG GTT CCGATG GAG-3′ (SEQ ID NO: 404) to introduce the random mutations in thePalm region. Brackets indicate a synthesis of 91% base shown and 3% allother bases.

[1148] 37. Construction of Tth AKK GL L422N/E425K (DNA Sequence SEQ IDNO:2741; Amino Acid Sequence SEQ ID NO:2742)

[1149] Palm region randomization mutagenesis was performed on the TthAKK GL construct using the mutagenic primer 680-79-02 5′-ctc cat cgg aacctc ctt [aag cgc ctc gag ggg gag gag aag ctc ctt tgg] ctc tac cac gaggtg-3′ (SEQ ID NO: 403) and reverse primer 680-79-04 5′-AAG GAG GTT CCGATG GAG-3′ (SEQ ID NO: 404) to introduce the random mutations in thePalm region. Brackets indicate a synthesis of 91% base shown and 3% allother bases.

[1150] 38. Construction of Tth AKK GL L422F/W430C (DNA Sequence SEQ IDNO:2743; Amino Acid Sequence SEQ ID NO:2744)

[1151] Palm region randomization mutagenesis was performed on Tth AKK GLDNA using the mutagenic primer 680-79-02 5′-ctc cat cgg aac ctc ctt [aagcgc ctc gag ggg gag gag aag ctc ctt tgg] etc tac cac gag gtg-3′ (SEQ IDNO: 403) and reverse primer 680-79-04 5′-AAG GAG GTT CCG ATG GAG-3′ (SEQID NO: 404) to introduce the random mutations in the Palm region.Brackets indicate a synthesis of 91% base shown and 3% all other bases.

[1152] 39. Construction of Tth AKK GL A504F (DNA Sequence SEQ IDNO:2745; Amino Acid Sequence SEQ ID NO:2746)

[1153] Site saturation mutagenesis was performed on Tth AKK GL constructusing the mutagenic primer 680-80-03 5′-cag gag ctt agg ctt ccc nnn ttgaag aag acg aag aag aca-3′ (SEQ ID NO: 413) and reverse primer 680-80-065′-cct aag ctc gtc aaa gag-3′ (SEQ ID NO: 414) to introduce the randommutations in the A504 amino acid.

[1154] 40. Construction of Tth AKK GL A504V (DNA Sequence SEQ IDNO:2747; Amino Acid Sequence SEQ ID NO:2748)

[1155] Site saturation mutagenesis was performed on Tth AKK GL constructusing the mutagenic primer 680-80-03 5′-cag gag ctt agg ctt ccc nnn ttgaag aag acg aag aag aca-3′ (SEQ ID NO: 413) and reverse primer 680-80-065′-cct aag ctc gtc aaa gag-3′ (SEQ ID NO: 414) to introduce the randommutations in the A504 amino acid.

[1156] 41. Construction of Tth AKK GL A504S (DNA Sequence SEQ IDNO:2749; Amino Acid Sequence SEQ ID NO: 2750)

[1157] Site saturation mutagenesis was performed on Tth AKK GL constructusing the mutagenic primer 680-80-03 5′-cag gag ctt agg ctt cc nnn ttgaag aag acg aag aag aca-3′ (SEQ ID NO: 413) and reverse primer 680-80-065′-cct aag ctc gtc aaa gag-3′ (SEQ ID NO: 414) to introduce the randommutations in the A504 amino acid.

[1158] 42. Construction of Tth AKK GL S517G (DNA Sequence SEQ IDNO:2751; Amino Acid Sequence SEQ ID NO: 2752)

[1159] Site saturation mutagenesis was performed on Tth AKK GL constructusing the mutagenic primer 680-80-07 5′-GGC AAG CGC TCC ACC NNN GCC GCGGTG CTG GAG GCC CTA CGG-3′ (SEQ ID NO: 421) and reverse primer 680-80-105′-GGT GGA GCG CTT GCC-3′ (SEQ ID NO: 422) to introduce the randommutations in the S517 amino acid.

[1160] 43 Construction of Tth AKK GL A518L (DNA Sequence SEQ ID NO:2753;Amino Acid Sequence SEQ ID NO:2754)

[1161] Site saturation mutagenesis was performed on Tth AKK GL constructusing the mutagenic primer 680-80-07 5′-GGC AAG CGC TCC ACC AGC NNN GCGGTG CTG GAG GCC CTA CGG-3′ (SEQ ID NO: 425) and reverse primer 680-80-105′-GGT GGA GCG CTT GCC-3′ (SEQ ID NO:422) to introduce the randommutations in the A518 amino acid.

[1162] 44. Construction of Tth AKK GL A518R (DNA Sequence SEQ IDNO:2755; Amino Acid Sequence SEQ ID NO:2756)

[1163] Site saturation mutagenesis was performed on Tth AKK GL constructusing the mutagenic primer 680-80-07 5′-GGC AAG CGC TCC ACC AGC NNN GCGGTG CTG GAG GCC CTA CGG-3′ (SEQ ID NO: 425) and reverse primer 680-80-105′-ggt gga gcg ctt gcc-3′ (SEQ ID NO: 422) to introduce the randommutations in the A518 amino acid.

[1164] 45. Construction of Tth AKK GL A504K (DNA Sequence SEQ IDNO:2757; Amino Acid Sequence SEQ ID NO:2758)

[1165] Site directed mutagenesis was performed on the Tth AKK GLconstruct using the mutagenic primer 680-69-04 5′-ctt agg ctt ccc aagttg aag aag acg aag aag aca-3′ (SEQ ID NO: 433) and reverse primer680-69-05 5′-tgt ctt ctt cgt ctt ctt caa ctt ggg aag cct aag-3′ (SEQ IDNO: 434) to introduce the A504K mutation in the Tth AKK GL enzyme.

[1166] 46. Construction of Tth AKK GL H641A (DNA Sequence SEQ IDNO:2759; Amino Acid Sequence SEQ ID NO:2760)

[1167] Site directed mutagenesis was performed on Tth AKK GL constructusing the mutagenic primer 680-69-08 5′-gag ggg aag gac atc gcc acc cagacc gca agc-3′ (SEQ ID NO: 437) and reverse primer 583-01-02 5′-gct tgcggt ctg ggt ggc gat gtc ctt ccc ctc-3′ (SEQ ID NO: 438) to introduce theH641A mutation in the Tth AKK GL enzyme.

[1168] 47. Construction of Tth AKK GL T508P (DNA Sequence SEQ IDNO:2761; Amino Acid Sequence SEQ ID NO:2762)

[1169] Site directed mutagenesis was performed on TthAKK GL constructusing the mutagenic primer 680-70-01 5′-ccc-gcc ttg aag aag ccg aag aagaca ggc aag-3′ (SEQ ID NO: 441) and reverse primer 680-70-02 5′-ctt gcctgt ctt ctt cgg ctt ctt caa ggc ggg-3′ (SEQ ID NO: 442) to introduce theT508P mutation in the Tth AKK GL enzyme.

[1170] 48. Construction of Taq(FT)TthAKK GL

[1171] Site specific mutagenesis was performed on the TaqTthAKK GLconstruct using the mutagenic primer 473-087-05:5′-cgg-gac-ctc-gag-gcg-cgt-gaa-ccc-cag-gag-gtc-cac-3′ (SEQ ID NO: 483)to introduce the L107F/A108T mutations and generate Taq(FT)TthAKK GL(DNA sequence SEQ ID NO:2779; amino acid sequence SEQ ID NO:2780).

[1172] 49. Construction of Tfi(FT)TthAKK GL

[1173] Site specific mutagenesis was performed on the TfiTthAKK GLconstruct using the mutagenic primers 785-096-01:5′-gtg-gac-ctt-ctg-ggc-ttt-acc-cgc-ctc-gag-gcc-ccg-3′ (SEQ ID NO: 486)and 785-096-02: 5′-cgg-ggc-ctc-gag-gcg-ggt-aaa-gcc-cag-aag-gtc-cac-3′(SEQ ID NO: 487) to introduce the L107F/V108T mutations and generateTfi(FT)TthAKK GL (DNA sequence SEQ ID NO:2720; amino acid sequence SEQID NO:2781).

[1174] 50. Construction of Tsc(FT)TthAKK GL

[1175] Site specific mutagenesis was performed on the TscTthAKK GLconstruct using the mutagenic primers 785-008-035′-ttt-acc-cgc-ctc-gag-gtg-ccg-ggc-3′ (SEQ ID NO: 489) and reverseprimer 680-21-03 5′-cgg cac ctc gag gcg ggt aaa gcc caa aag gtc cac-3′(SEQ ID NO: 490) to introduce the L107F/E108T mutations and generateTsc(FT)TthAKK GL (DNA sequence SEQ ID NO:2764; amino acid sequence SEQID NO:2782).

[1176] 51. Fusion Between TthAKK GL Enzyme and Alpha-Peptide

[1177] A TthAKK GL-lacZ-alpha-peptide chimeric fusion was constructed toallow detection of mutations (including frame-shifts, deletions,insertions, etc.) which cause the inability of expression of thefull-length fusion protein based on the colony blue-white screening (Wuet al., Nucleic Acids Research, 24:1710 [1996]).

[1178] Site specific mutagenesis was performed on TthAKK GL DNA usingthe mutagenic primers 959-041-03,5′-cac-cac-cac-cac-cac-cac-gtc-gac-tag-tgc-tag-cgt-cga-cta-gct-gca-ggc-atg-caa-gct-tgg-c-3′(SEQ ID NO: 477) and 959-041-04,5′-gcc-aag-ctt-gca-tgc-ctg-cag-cta-gtc-gac-gct-agc-act-agt-cga-cgt-ggt-ggt-ggt-ggt-ggt-g-3′(SEQ ID NO: 478) to generate pTthAKK GL-L with SalI site following the6×His tag at the C-terminus of TthAKK GL for the insertion of lacZ alphapeptide. The alpha peptide (of 201 amino acids) was first PCR amplifiedfrom the pCRII-TOPO vector (Invitrogen) with primers 959-041-01,5′-cag-gaa-gcg-gcc-gcg-tcg-aca-tga-cca-tga-tta-cgc-caa-gc-3′ (SEQ ID NO:479) and 959-093-01,5′-ggg-ccc-gcc-agg-gtc-gac-tca-ggg-cga-tgg-ccc-act-acg-tga-3′ (SEQ IDNO: 480). The PCR product was then digested with restriction enzyme SalIand ligated into the pTthAKK GL-L vector, which was also cut with thesame enzyme to generate the chimeric construct TthAKK GL-alpha peptide(DNA sequence SEQ ID NO:2777; amino acid sequence SEQ ID NO:2778). Theorientation of the insert was confirmed by sequencing.

[1179] 52. Construction of TaqEFT-Tth(AKK) GL (DNA Sequence SEQ IDNO:2790; Amino Acid Sequence SEQ ID NO:2791)

[1180] Site specific mutagenesis was performed on Taq(FT)-Tth(AKK) GLDNA (SEQ ID NO: 2779) using the mutagenic primers 436-013-08:5′-atc-gtg-gtc-ttt-gac-gcc-gag-gcc-ccc-tcc-ttc-c-3′ (SEQ ID NO:503) and436-013-09 5′-gga-agg-agg-ggg-cct-cgg-cgt-caa-aga-cca-cga-t-3′ (SEQ IDNO:504) to introduce the K70E mutation.

[1181] 53. Construction of TaqEFT-Tth(AKK) GL-A/M1 (DNA Sequence SEQ IDNO:2792; Amino Acid Sequence SEQ ID NO:2793)

[1182] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) GLDNA (SEQ ID NO:2790) using the mutagenic primers 1044-038-01:5′-cag-acc-atg-aat-tcg-gag-gcg-atg-ctg-ccc-ctc-ttt-3′ (SEQ ID NO:507)and 1044-038-02: (SEQ ID NO: 508)5′-aaa-gag-ggg-cag-cat-cgc-ctc-cga-att-cat-ggt-ctg-3′ to introduce theG4EA mutation.

[1183] 54. Construction of TaqEFT-Tth(AKK) GL-B/M2 (DNA Sequence SEQ IDNO:2794; Amino Acid Sequence SEQ ID NO: 2795)

[1184] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) GLDNA (SEQ ID NO: 2790) using the mutagenic primers 1044-038-03:5′-gcc-tac-cgc-acc-ttc-ttt-gcc-ctg-aag-ggc-ctc-3 and 1044-038-04:5′-gag-gcc-ctt-cag-ggc-aaa-gaa-ggt-gcg-gta-ggc-3′ (SEQ ID NO: 511 and512, respectively) to introduce the H₂₉F mutation.

[1185] 55. Construction of TaqEFT-Tth(AKK) GL-C/M3 (DNA Sequence SEQ IDNO:2796; Amino Acid Sequence SEQ ID NO:2797)

[1186] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) GLDNA (SEQ ID NO: 2790) using the mutagenic primers 1044-038-05:5′-ctc-ctc-aag-gcc-ctc-aga-gag-gac-ggg-gac-gcg-3′ and 1044-038-06:5′-cgc-gtc-ccc-gtc-ctc-tct-gag-ggc-ctt-gag-gag-3′ (SEQ ID NO: 515 and516, respectively) to introduce the K57R mutation.

[1187] 56. Construction of TaqEFT-Tth(AKK) GL-DIM5 (DNA Sequence SEQ IDNO:2798; Amino Acid Sequence SEQ ID NO:2799)

[1188] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) GLDNA (SEQ ID NO: 2790) using the mutagenic primers 1044-038-09:5′-gac-gac-gtc-ctg-gcc-acc-ctg-gcc-aag-aag-gcg-3′ and 1044-038-10:5′-cgc-ctt-ctt-ggc-cag-ggt-ggc-cag-gac-gtc-gtc-3′ (SEQ ID NO: 519 and520, respectively) to introduce the S125T mutation.

[1189] 57. Construction of TaqEFT-Tth(AKK) GL-E/M6 (DNA Sequence SEQ IDNO:2800; Amino Acid Sequence SEQ ID NO:2801)

[1190] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) GLDNA (SEQ ID NO: 2790) using the mutagenic primers 1044-038-11:5′-ggg-gag-aag-acg-gcg-ctc-aag-ctt-ctg-gag-gag-3′ and 1044-038-12:5′-ctc-ctc-cag-aag-ctt-gag-cgc-cgt-ctt-ctc-ccc-3′ (SEQ ID NO: 523 and524, respectively) to introduce the R206L mutation.

[1191] 58. Construction of TaqEFT-Tth(AKK) GL-F/M7 (DNA Sequence SEQ IDNO:2802; Amino Acid Sequence SEQ ID NO:2803)

[1192] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) GLDNA (SEQ ID NO:2790) using the mutagenic primers 1044-038-13:5′-gag-ccc-gac-cgg-gag-ggg-ctt-aag-gcc-ttt-ctg-gag-agg-3′ and1044-038-14: 5′-cct-ctc-cag-aaa-ggc-ctt-aag-ccc-ctc-ccg-gtc-ggg-ctc-3(SEQ ID NO: 527 and 528, respectively) to introduce the R269G and R271Kmutations.

[1193] 59. Construction of TaqEFT-Tth(AKK) GL-G/M8 (DNA Sequence SEQ IDNO:2804; Amino Acid Sequence SEQ ID NO:2805)

[1194] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) GLDNA (SEQ ID NO: 2790) using the mutagenic primers 1044-038-15:5′-cac-gag-ttc-ggc-ctt-ctg-gga-ggg-gag-aag-ccc-cgg-gag-gag-gcc-ccc-tgg-ccc-3′and 1044-038-16:5′-ggg-cca-ggg-ggc-ctc-ctc-ccg-ggg-ctt-ctc-ccc-tcc-cag-aag-gcc-gaa-ctc-gtg-3′(SEQ ID NO: 531 and 532, respectively) to introduce the E290G, S291G,P292E, A294P, and L295R mutations.

[1195] 60. Construction of TaqEFT-Tth(AKK) GL-H/M9 (DNA Sequence SEQ IDNO:2806; Amino Acid Sequence SEQ ID NO:2807)

[1196] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) GLDNA (SEQ ID NO: 2790) using the mutagenic primers 1044-038-17:5′-ctg-gcc-ctg-gcc-gcc-tgc-agg-ggc-ggc-cgc-gtg-3′ and 1044-038-18:5′-cac-gcg-gcc-gcc-cct-gca-ggc-gge-cag-ggc-cag-3′ (SEQ ID NO: 535 and536, respectively) to introduce the A328C mutation.

[1197] 61. Construction of TaqEFT-Tth(AKK) GL-I/M10 (DNA Sequence SEQ IDNO:2808; Amino Acid Sequence SEQ ID NO:2809)

[1198] Site specific mutagenesis was performed on Taq(EFT)-Tth(AKK) GLDNA (SEQ ID NO: 2790) using the mutagenic primers 1080-015-01 (SEQ IDNO:539) 5′-ggg gag aag acg gcg agg aag ctt ctg aag gag tgg ggg agc-3′and 1080-015-02 (SEQ ID NO: 540) 5′-gct ccc cca ctc ctt cag aag ctt cctcgc cgt ctt ctc ccc-3′ to introduce the E21 OK mutation.

[1199] 62. Construction of TaqEFT-Tth(AKK) GL-Ml-9 (DNA Sequence SEQ IDNO:2810; Amino Acid Sequence SEQ ID NO:2811)

[1200] Seven independent PCR reactions were performed, using constructTaqEFT-Tth(AKK) GL (SEQ ID: 2790) as a template, with the followingpairs of mutagenic primers: PCR Reaction 1; 1044-038-015′-cag-acc-atg-aat-tcg-gag-gcg-atg-ctg-ccc-ctc-ttt-3′ (SEQ ID NO:507)and 1044-038-04 5′-gag-gcc-ctt-cag-ggc-aaa-gaa-ggt-gcg-gta-ggc-3′ (SEQID NO:512) to yield a 108 base pair fragment, PCR Reaction 2;1044-038-03 5′-gcc-tac-cgc-acc-ttc-ttt-gcc-ctg-aag-ggc-ctc-3′ (SEQ IDNO: 511) and 1044-038-065′-cgc-gtc-ccc-gtc-ctc-tct-gag-ggc-ctt-gag-gag-3′ (SEQ ID NO: 516) toyield a 117 base pair fragment, PCR Reaction 3; 1044-038-055′-ctc-ctc-aag-gcc-ctc-aga-gag-gac-ggg-gac-gcg-3′ (SEQ ID NO: 515) and1044-038-10 5′-cgc-ctt-ctt-ggc-cag-ggt-ggc-cag-gac-gtc-gtc-3′ (SEQ IDNO: 520) to yield a 237 base pair fragment, PCR Reaction 4; 1044-038-095′-gac-gac-gtc-ctg-gcc-acc-ctg-gcc-aag-aag-gcg-3′ (SEQ ID NO: 519) and1044-038-12 5′-ctc-ctc-cag-aag-ctt-gag-cgc-cgt-ctt-ctc-ccc-3′ (SEQ IDNO: 524) to yield a 276 base pair fragment, PCR Reaction 5; 1044-038-115′-ggg-gag-aag-acg-gcg-ctc-aag-ctt-ctg-gag-gag-3′ (SEQ ID NO: 523) and1044-038-14 5′-cct-ctc-cag-aaa-ggc-ctt-aag-ccc-ctc-ccg-gtc-ggg-ctc-3′(SEQ ID NO: 528) to yield a 228 base pair fragment, PCR Reaction 6;1044-038-13 5′-gag-ccc-gac-cgg-gag-ggg-ctt-aag-gcc-ttt-ctg-gag-agg-3′(SEQ ID NO: 527) and 1044-038-165′-ggg-cca-ggg-ggc-ctc-ctc-ccg-ggg-ctt-ctc-ccc-tcc-cag-aag-gcc-gaa-ctc-gtg-3′(SEQ ID NO: 532) to yield a 113 base pair fragment, PCR Reaction 7;1044-038-155′-cac-gag-ttc-ggc-ctt-ctg-gga-ggg-gag-aag-ccc-cgg-gag-gag-gcc-ccc-tgg-ccc-3′(SEQ ID NO: 531) and 1044-038-185′-cac-gcg-gcc-gcc-cct-gca-ggc-ggc-cag-ggc-cag-3′ (SEQ ID NO: 536) toyield a 157 base pair fragment. The seven PCR products overlap such thatPCR amplification in the absence of primers yielded the appropriate 1005base pair product to introduce the K70E, G4EA, H₂₉F, K57R, S125T, R206L,R269G, R271K, E290G, S291G, P292E, A294P, L295R, and A328C mutations.The product was further amplified using the outside primers: 1044-038-1(SEQ ID NO: 507) and 1044-038-18 (SEQ ID NO: 536) and cloned intopPCR-Script-Amp. From this construct, the nuclease domain was againamplified using primers: 1044-038-1 (SEQ ID NO: 507) and 1044-038-18(SEQ ID NO: 536) and digested with EcoRI and NotI. The digested PCRproduct was ligated into an EcoRI and NotI digested TaqEFT-Tth(AKK) GLconstruct and transformed into JM109 for protein expression andscreening.

[1201] 63. Construction of TaqEFT-Tth(AKK) GL-Ml-10 (DNA Sequence SEQ IDNO:2812; Amino Acid Sequence SEQ ID NO:2813)

[1202] Seven independent PCR reactions were performed, using constructTaqEFT-Tth(AKK) GL (SEQ ID: 2790) as a template, with the followingpairs of mutagenic primers: PCR Reaction 1; 1044-038-015′-cag-acc-atg-aat-tcg-gag-gcg-atg-ctg-ccc-ctc-ttt-3′ (SEQ ID NO:507)and 1044-038-04 5′-gag-gcc-ctt-cag-ggc-aaa-gaa-ggt-gcg-gta-ggc-3′ (SEQID NO:512) to yield a 108 base pair fragment, PCR Reaction 2;1044-038-03 5′-gcc-tac-cgc-acc-ttc-ttt-gcc-ctg-aag-ggc-ctc-3′ (SEQ IDNO: 511) and 1044-038-065′-cgc-gtc-ccc-gtc-ctc-tct-gag-ggc-ctt-gag-gag-3′ (SEQ ID NO: 516) toyield a 117 base pair fragment, PCR Reaction 3; 1044-038-055′-ctc-ctc-aag-gcc-ctc-aga-gag-gac-ggg-gac-gcg-3′ (SEQ ID NO: 515) and1044-038-10 5′-cgc-ctt-ctt-ggc-cag-ggt-ggc-cag-gac-gtc-gtc-3′ (SEQ IDNO: 520) to yield a 237 base pair fragment, PCR Reaction 4; 1044-038-095′-gac-gac-gtc-ctg-gcc-acc-ctg-gcc-aag-aag-gcg-3′ (SEQ ID NO: 519) and1080-42-02 5′-gc ttc cag gct ccc cca ctc ctt cag aag ctt gag cgc cgt cttctc ccc-3′ (SEQ ID NO: 546) to yield a 299 base pair fragment, PCRReaction 5; 1080-42-01 5′-ggg gag aag acg gcg ctc agg ctt ctg aag gagtgg ggg agc ctg gaa gc-3′ (SEQ ID NO: 545) and 1044-038-145′-cct-ctc-cag-aaa-ggc-ctt-aag-ccc-ctc-ccg-gtc-ggg-ctc-3′ (SEQ ID NO:528) to yield a 228 base pair fragment, PCR Reaction 6; 1044-038-135′-gag-ccc-gac-cgg-gag-ggg-ctt-aag-gcc-ttt-ctg-gag-agg-3′ (SEQ ID NO:527) and 1044-038-165′-ggg-cca-ggg-ggc-ctc-ctc-ccg-ggg-ctt-ctc-ccc-tcc-cag-aag-gcc-gaa-ctc-gtg-3′(SEQ ID NO: 532) to yield a 113 base pair fragment, PCR Reaction 7;1044-038-155′-cac-gag-ttc-ggc-ctt-ctg-gga-ggg-gag-aag-ccc-cgg-gag-gag-gcc-ccc-tgg-ccc-3′(SEQ ID NO: 531) and 1044-038-185′-cac-gcg-gcc-gcc-cct-gca-ggc-ggc-cag-ggc-cag-3′ (SEQ ID NO: 536) toyield a 157 base pair fragment. The seven PCR products overlap such thatPCR amplification in the absence of primers yielded the appropriate 1005base pair product to introduce the K70E, G4EA, H₂₉F, K57R, S125T, R206L,E210K, R269G, R271K, E290G, S291G, P292E, A294P, L295R, and A328Cmutations. The product was further amplified using the outside primers:1044-038-1 (SEQ ID NO: 507) and 1044-038-18 (SEQ ID NO: 536) and clonedinto pPCR-Script-Amp. From this construct, the nuclease domain was againamplified using primers: 1044-038-1 (SEQ ID NO: 507) and 1044-038-18(SEQ ID NO: 536) and digested with EcoRI and NotI. The digested PCRproduct was ligated into an EcoRI and NotI digested TaqEFT-Tth(AKK) GLconstruct and transformed into JM109 for enzyme expression andscreening.

[1203] 64. Construction of Tth(K69E)AKK, Taq(K69E)TthAKK,Tfi(K69E)TthAKK and Tsc(K69E)TthAKK and Mutants

[1204] Site specific mutagenesis was performed on TthAKK GL, TaqTthAKKGL, TfiTthAKK GL and TscTthAKK GL DNAs using the mutagenic primers5′-atc-gtg-gtc-ttt-gac-gcc-gag-gcc-ccc-tcc-ttc-c-3′ (SEQ ID NO: 492) and5′-gga-agg-agg-ggg-cct-cgg-cgt-caa-aga-cca-cga-t-3′ (SEQ ID NO: 493) tointroduce the K69E mutation and to generate Tth(K69E)AKK GL (DNAsequence SEQ ID NO:2763; amino acid sequence SEQ ID NO:2783),Taq(K69E)ThAKK GL, (DNA sequence SEQ ID NO:2784; amino acid sequence SEQID NO:2785), Tfi(K69E)TthAKK GL (DNA sequence SEQ ID NO:2786; amino acidsequence SEQ ID NO:2787) and Tsc(K69E)TthAKK GL (DNA sequence SEQ IDNO:2788; amino acid sequence SEQ ID NO:2789) mutant enzymes.

[1205] 65. Construction of Tsc(167-334)TthAKK GL

[1206] Two overlapping PCR fragments were generated by primers390-76-08: 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-041-01:5′-ctt-ctc-tca-tcc-gcc-aaa-aca-gcc-3′ (SEQ ID NO: 451) with templateTth(K69E)AKK GL, (DNA sequence SEQ ID NO:2763), and primers 1044-041-02:5′-ctc-ctc-cac-gag-ttc-ggc-3′ (SEQ ID NO: 453) and 209-074-02:5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) with templateTsc(K69E)TthAKK GL, (DNA sequence SEQ ID NO:2764). Two products werecombined and amplified with outside primers 390-76-08 and 209-074-02.The recombinant PCR product was cut with the restriction enzymes EcoRIand NotI and ligated into the vector TthAKK GL which was prepared bycutting with the same enzymes to yield Tsc(167-333)TthAKK GL construct(DNA sequence SEQ ID NO:2765; amino acid sequence SEQ ID NO:2766).

[1207] 66. Construction of Tsc(222-334)TthAKK GL

[1208] Two overlapping PCR fragments were generated by primers390-76-08: 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-041-03:5′-ttc-cag-gtg-ctt-gag-gag-gtt-ttc-cag-3′ (SEQ ID NO: 457) with templateTth(K69E)AKK GL (DNA sequence SEQ ID NO:2763), and primers 1044-041-04:5′-ctc-ctc-aag-cac-ctg-gaa-cag-gtg-aaa-3′ (SEQ ID NO: 458) and209-074-02: 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) withtemplate Tsc(K69E)TthAKK GL, (DNA sequence SEQ ID NO:2764). Two productswere combined and amplified with outside primers 390-76-08 and209-074-02. The recombinant PCR product was cut with the restrictionenzymes EcoRI and NotI and ligated into the vector TthAKK GL which wasprepared by cutting with the same enzymes to yield Tsc(222-334)TthAKK GLconstruct (DNA sequence SEQ ID NO:2767; amino acid sequence SEQ IDNO:2768).

[1209] 67. Construction of Tfi(222-334)TthAKK GL

[1210] Two overlapping PCR fragments were generated by primers390-76-08: 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-058-05:5′-gtc-cag-gtt-ctt-gag-gag-gtt-ttc-cag-3′ (SEQ ID NO: 461) with templateTth(K69E)AKK GL (DNA sequence SEQ ID NO:2763), and primers 1044-058-06:5′-ctc-ctc-aag-aac-ctg-gac-cgg-gta-aag-3′ (SEQ ID NO: 462) and209-074-02: 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) withtemplate Tfi(K69E)TthAKK GL (DNA sequence SEQ ID NO:2786). Two productswere combined and amplified with outside primers 390-76-08 and209-074-02. The recombinant PCR product was cut with the restrictionenzymes EcoRI and NotI and ligated into the vector TthAKK GL which wasprepared by cutting with the same enzymes to yield Tfi(222-334)TthAKK GLconstruct (DNA sequence SEQ ID NO:2769; amino acid sequence SEQ IDNO:2770).

[1211] 68. Construction of Tfi(167-334)TthAKK GL

[1212] Two overlapping PCR fragments were generated by primers 390-76-085′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-058-075′-gac-gtc-ctt-cgg-ggt-gat-gag-gtg-gcc-3′ (SEQ ID NO: 465) with templateTth(K69E)AKK GL, (DNA sequence SEQ ID NO:2763), and primers 1044-058-085′-atc-acc-ccg-aag-gac-gtc-cag-gag-aag-3′ (SEQ ID NO: 466) and209-074-02 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) withtemplate Tfi(K69E)TthAKK GL, (DNA sequence SEQ ID NO:2787). Two productswere combined and amplified with outside primers 390-76-08 and209-074-02. The recombinant PCR product was cut with the restrictionenzymes EcoRI and NotI and ligated into the vector TthAKK GL which wasprepared by cutting with the same enzymes to yield Tfi(167-333)TthAKK GLconstruct (DNA sequence SEQ ID NO: 2771; amino acid sequence SEQ IDNO:2772).

[1213] 69. Construction of Tsc(111-334)TthAKK GL

[1214] Two overlapping PCR fragments were generated by primers 390-76-085′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-058-015′-ctc-gag-gcg-ggt-aaa-ccc-cag-gag-gtc-3′ (SEQ ID NO: 469) with templateTth(K69E)AKK GL (DNA sequence SEQ ID NO:2763), and primers 1044-058-025′-ggg-ttt-acc-cgc-ctc-gag-gtg-ccc-ggc-3′ (SEQ ID NO: 470) and209-074-02 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) withtemplate Tsc(K69E)TthAKK GL (DNA sequence SEQ ID NO:2788). Two productswere combined and amplified with outside primers 390-76-08 and209-074-02. The recombinant PCR product was cut with the restrictionenzymes EcoRI and NotI and ligated into the vector TthAKK GL which wasprepared by cutting with the same enzymes to yield Tsc(167-333)TthAKK GLconstruct (DNA sequence SEQ ID NO:2773; amino acid sequence SEQ IDNO:2774).

[1215] 70. Construction of Tsc(1-167)TthAKK GL

[1216] Two overlapping PCR fragments were generated by primers 390-76-085′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and 1044-058-035′-aag-cca-ctc-cgg-ggt-gat-cag-gta-acc-3′ (SEQ ID NO: 473) with templateTsc(K69E)TthAKK GL (DNA sequence SEQ ID NO:2788), and primers1044-058-04 5′-atc-acc-ccg-gag-tgg-ctt-tgg-gag-aag-3′ (SEQ ID NO: 474)and 209-074-02 5′-gtg-gcc-tcc-ata-tgg-gcc-agg-ac-3′ (SEQ ID NO:316) withtemplate Tth(K69E)AKK GL (DNA sequence SEQ ID NO:2763). Two productswere combined and amplified with outside primers 390-76-08 and209-074-02. The recombinant PCR product was cut with the restrictionenzymes EcoRI and NotI and ligated into the vector TthAKK GL which wasprepared by cutting with the same enzymes to yield Tsc(1-167)TthAKK GLconstruct (DNA sequence SEQ ID NO:2775; amino acid sequence SEQ IDNO:2776).

[1217] 71. Construction of Afu336-Tth296(AKK) GL, AT1-1

[1218] Two overlapping PCR fragments were generated. The first fragmentwas made using the AfuFEN construct (SEQ ID NO:556) as template with theprimers: 390-076-08 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and390-065-05 5′-gaa-cca-cct-ctc-aag-cgt-gg-3′ (SEQ ID NO: 554). The secondfragment was made using TthAKK GL (SEQ ID NO:2702) as template and theprimers: 700-049-01 5′-acg-ctt-gag-agg-tgg-ttc-ctg-gag-gag-gc-ccc-tgg-3′(SEQ ID NO: 555) and 390-076-09 5′-taa-tct-gta-tca-ggc-tg-3′ (SEQ IDNO:557). The two products contain a region of sequence overlap, and werecombined and amplified with outside primers 390-076-08 and 390-076-09 ina recombinant PCR reaction. The recombinant PCR product was cut with therestriction enzymes BspEI and SalI and ligated into the AfuFEN constructwhich was prepared by cutting with the same enzymes to yieldAfu336-Tth296(AKK) GL construct (DNA sequence SEQ ID NO:2814; amino acidsequence SEQ ID NO:2815).

[1219] 72. Construction of Afu328-Tth296(AKK) GL, AT2-3

[1220] Two overlapping PCR fragments were generated, the first byprimers: 390-076-08 5′-tgt-gga-att-gtg-agc-gg-3′ (SEQ ID NO:314) and700-049-02 5′-ggt-tga-ctt-cag-agc-ttt-gag-3′ (SEQ ID NO: 561) withtemplate AfuFEN (DNA sequence SEQ ID NO:556), and the second by primers:700-049-03 5′-aaa-gct-ctg-aag-tca-acc-ctg-gag-gag-gcc-ccc-tgg-3′ (SEQ IDNO: 562) and 390-076-09 5′-taa-tct-gta-tca-ggc-tg-3′ (SEQ ID NO:557)with template TthAKK GL (DNA sequence SEQ ID NO:2702). The two productswere combined and amplified with outside primers 390-076-08 and390-076-09. The recombinant PCR product was cut with the restrictionenzymes BspEI and SalI and ligated into the vector pAfuFEN which wasprepared by cutting with the same enzymes to yield Afu336-Tth296(AKK) GLconstruct (DNA sequence SEQ ID NO:2816; amino acid sequence SEQ IDNO:2817).

[1221] The procedure for random chimerization desribed above was used todevelop the following chimeras.

[1222] 73. Generation of Random Chimeras S26 and S36

[1223] The templates used to develop these chimeric enzymes were thenuclease domains from TthAKK GL, TaqTthAKK GL, TscTthAKK GL and theTfiTthAKK GL constructs. Two clones were found to show improvement ofactivity based on primary activity screening. Random chimeras S26 GL(DNA sequence SEQ ID NO:2818; amino acid sequence SEQ ID NO:2819) andS36 (DNA sequence SEQ ID NO:2820; amino acid sequence SEQ ID NO:2821)were then sequenced and isolated.

[1224] 74. Construction of S26(FT) GL

[1225] Site specific mutagenesis was performed on pS26 GL DNA using themutagenic primers: 785-008-03 5′-ttt-acc-cgc-ctc-gag-gtg-ccg-ggc-3′ (SEQID NO: 489) and 680-21-035′-cgg-cac-ctc-gag-gcg-ggt-aaa-gcc-caa-aag-gtc-cac-3′ (SEQ ID NO: 490)to introduce the L107F/E108T mutations and generate S26(FT) GL (DNAsequence SEQ ID NO:2822; amino acid sequence SEQ ID NO:2823).

[1226] 75. Construction of S36(FT) GL

[1227] Site specific mutagenesis was performed on pS36 GL DNA using themutagenic primers: 785-096-01:5′-gtg-gac-ctt-ctg-ggc-ttt-acc-cgc-ctc-gag-gcc-ccg-3′ (SEQ ID NO: 486)and 785-096-02: 5′-cgg-ggc-ctc-gag-gcg-ggt-aaa-gcc-cag-aag-gtc-cac-3′(SEQ ID NO: 487) to introduce the L109F/V110T mutations and generateS36(FT) GL (DNA sequence SEQ ID NO: 2824; amino acid sequence SEQ IDNO:2825).

[1228] 76. Construction of S26(K69E) GL

[1229] Site specific mutagenesis was performed on S26 GL DNA using themutagenic primers: 5′-atc-gtg-gtc-ttt-gac-gcc-gag-gcc-ccc-tcc-ttc-c-3′(SEQ ID NO: 492) and 5′-gga-agg-agg-ggg-cct-cgg-cgt-caa-aga-cca-cga-t-3′(SEQ ID NO: 493) to introduce the K69E mutation and to generateS26(K69E) GL (DNA sequence SEQ ID NO:2826; amino acid sequence SEQ IDNO:2827).

[1230] 77. Construction of S26(FT/K69E) GL

[1231] Site specific mutagenesis was performed on S26(FT) GL DNAs usingthe mutagenic primers:5′-atc-gtg-gtc-ttt-gac-gcc-gag-gcc-ccc-tcc-ttc-c-3′ (SEQ ID NO: 492) and5′-gga-agg-agg-ggg-cct-cgg-cgt-caa-aga-cca-cga-t-3′ (SEQ ID NO: 493) tointroduce the K69E mutation and to generate S26(FT/K69E) GL (DNAsequence SEQ ID NO:2828; amino acid sequence SEQ ID NO:2829).

[1232] 78. More Random Chimeras, N3D7, N1A12 N1C4, and N2C3

[1233] The templates used to generate these chimerics were the nucleasedomains of Tth(K69E)AKK GL, Taq(K69E)TthAKK GL, Tsc(K69E)TthAKK GL andTfi(K69E)TthAKK GL constructs. Four clones were shown to have improvedactivity based on the primary activity screening. Random chimeras N3D7GL (DNA sequence SEQ ID NO:2830; amino acid sequence SEQ ID NO:2831),N1A12 GL (DNA sequence SEQ ID NO:2832; amino acid sequence SEQ IDNO:2833), N1C4 GL (DNA sequence SEQ ID NO:2834; amino acid sequence SEQID NO:2835) and N2C3 GL (DNA sequence SEQ ID NO:2836; amino acidsequence SEQ ID NO:2837) were then sequenced and isolated respectively.

[1234] Generation of DL, GR and DR Variants of TthAKK GL Enzyme

[1235] A series of TthAKK GL variants having a Gly to Asp change atamino acid 385 (G385D) and/or a Leu to Arg change at amino acid 455(L455R), were created by site-directed mutagenesis. Constructscomprising both of these two amino acid variations is indicated as “DR”.Chimeras comprising only one variation are indicated as either ‘DL’ or‘GR’.

[1236] 1. Construction of TthAKK DL Mutant

[1237] Site specific mutagenesis was performed on pTrc99A TthAKK GL DNA(SEQ ID NO: 2702) using the mutagenic primers 1144-60-01,5′-cgc-cta-cct-cct-gga-ccc-ttc-gaa-cac-cac-c-3′ (SEQ ID NO:2858) and1144-60-02, 5′-ggt-ggt-gtt-cga-agg-gtc-cag-gag-gta-ggc-g-3′ (SEQ IDNO:2859) to introduce the G383D mutation to yield TthAKK DL (DNAsequence SEQ ID NO:2838; amino acid sequence SEQ ID NO:2839).

[1238] 2. Construction of TthAKK GR Mutant

[1239] Site specific mutagenesis was performed on pTrc99A TthAKK GL DNA(SEQ ID NO: 2702) using the mutagenic primers 1144-60-03,5′-ggt-acg-gcg-gga-cgt-ggc-cta-cct-tca-g-3′ (SEQ ID NO:2860) and1144-60-04, 5′-ctg-aag-gta-ggc-cac-gtc-ccg-ccg-tac-c-3′ (SEQ ID NO:2861)to introduce the L453R mutation to yield TthAKK GR (DNA sequence SEQ IDNO:2840; amino acid sequence SEQ ID NO:2841).

[1240] 3. Construction of TthAKK DR Mutant

[1241] Site specific mutagenesis was performed on pTrc99A TthAKK GL DNA(SEQ ID NO: 2702) using the mutagenic primers 1144-60-01, 1144-60-02,1144-60-03 and 1144-60-04 (SEQ ID NOS:2858, 2859, 2860 and 2861,respectively) to introduce the G383D and L453R mutations to yield TthAKKDR mutant (DNA sequence SEQ ID NO:2842; amino acid sequence SEQ IDNO:2843).

[1242] 4. Construction of TthAKK(F29R) GL

[1243] Site specific mutagenesis was performed on pTrc99A TthAKK GL DNA(SEQ ID NO: 2702) using the mutagenic primers 1249-26-01,5′-cac-ctg-gcc-tac-cgc-acc-cgc-ttc-gcc-ctg-aag-ggc-ctc-3′ (SEQ IDNO:2862) and 1249-26-02,5′-gag-gcc-ctt-cag-ggc-gaa-gcg-ggt-gcg-gta-ggc-cag-gtg-3′ (SEQ IDNO:2863) to introduce the F29R mutation to yield the TthAKK(F29R) GLmutant (DNA sequence SEQ ID NO:2844; amino acid sequence SEQ IDNO:2845).

[1244] 5. Construction of TthAKK(F29R/F30H) GL

[1245] Site specific mutagenesis was performed on pTrc99A TthAKK GL DNA(SEQ ID NO: 2702) using the mutagenic primers 1249-26-03,5′-cac-ctg-gcc-tac-cgc-acc-cgc-cac-gcc-ctg-aag-ggc-ctc-acc-3′ (SEQ IDNO:2864) and 1249-26-04,5′-ggt-gag-gcc-ctt-cag-ggc-gtg-gcg-ggt-gcg-gta-ggc-cag-gtg-3′ (SEQ IDNO:2865) to introduce the F29R/F30H mutations to yield theTthAKK(F29R/F30H) GL mutant (DNA sequence SEQ ID NO:2846; amino acidsequence SEQ ID NO:2847).

[1246] 6. Construction of TthAKK(F29R/F30R) GL

[1247] Site specific mutagenesis was performed on pTrc99A TthAKK GL DNA(SEQ ID NO: 2702) using the mutagenic primers 1249-26-05,5′-cac-ctg-gcc-tac-cgc-acc-cgc-cgc-gcc-ctg-aag-ggc-ctc-acc-3′ (SEQ IDNO:2866) and 1249-26-06,5′-ggt-gag-gcc-ctt-cag-ggc-gcg-gcg-ggt-gcg-gta-ggc-cag-gtg-3′ (SEQ IDNO:2867) to introduce the F29R/F30R mutations to yield theTthAKK(F29R/F30R) GL mutant (DNA sequence SEQ ID NO:2848; amino acidsequence SEQ ID NO:2849).

[1248] 7. Construction of Tsc(F26R)TthAKK GL

[1249] Site specific mutagenesis was performed on pTrc99A TscTth(AKK) GLDNA (SEQ ID NO: 2708) using the mutagenic primers 1249-26-01,5′-cac-ctg-gcc-tac-cgc-acc-cgc-tc-gcc-ctg-aag-ggc-ctc-3′ (SEQ IDNO:2862) and 1249-26-02,5′-gag-gcc-ctt-cag-ggc-gaa-gcg-ggt-gcg-gta-ggc-cag-gtg-3′ (SEQ IDNO:2863) to introduce the F26R mutation to yield the Tsc(F26R)TthAKK GLmutant (DNA sequence SEQ ID NO:2850; amino acid sequence SEQ IDNO:2851).

[1250] 8. Construction of Tsc(F26R/F27H)TthAKK GL

[1251] Site specific mutagenesis was performed on pTrc99A TscTthAKK GLDNA (SEQ ID NO: 2708) using the mutagenic primers 1249-26-03,5′-cac-ctg-gcc-tac-cgc-acc-cgc-cac-gcc-ctg-aag-ggc-ctc-acc-3′ (SEQ IDNO:2864) and 1249-26-04,5′-ggt-gag-gcc-ctt-cag-ggc-gtg-gcg-ggt-gcg-gta-ggc-cag-gtg-3′ (SEQ IDNO:2865) to introduce the F26R/F27H mutations to yield theTsc(F26R/F27H)TthAKK GL mutant (DNA sequence SEQ ID NO:2852; amino acidsequence SEQ ID NO:2853).

[1252] 9. Construction of Tsc(F26R/F27R)TthAKK GL

[1253] Site specific mutagenesis was performed on pTrc99A TscTthAKK GLDNA (SEQ ID NO: 2708) using the mutagenic primers 1249-26-05,5′-cac-ctg-gcc-tac-cgc-acc-cgc-cgc-gcc-ctg-aag-ggc-ctc-acc-3′ (SEQ IDNO:2866) and 1249-26-06,5′-ggt-gag-gcc-ctt-cag-ggc-gcg-gcg-ggt-gcg-gta-ggc-cag-gtg-3′ (SEQ IDNO:2867) to introduce the F26R/F27R mutations to yield theTsc(F26R/F27R)TthAKK GL mutant (DNA sequence SEQ ID NO:2854; amino acidsequence SEQ ID NO:2855).

[1254] 10. Construction of Taq(F28R)TthAKK GL

[1255] Site specific mutagenesis was performed on pTrc99A-TaqTthAKK GLDNA (SEQ ID NO: 2706) using the mutagenic primers 1249-26-03,5′-cac-ctg-gcc-tac-cgc-acc-cgc-cac-gcc-ctg-aag-ggc-ctc-acc-3′ (SEQ IDNO:2864) and 1249-26-04,5′-ggt-gag-gcc-ctt-cag-ggc-gtg-gcg-ggt-gcg-gta-ggc-cag-gtg-3′ (SEQ IDNO:2865) to introduce the F28R mutation and generate Taq(F28R)TthAKK GLmutant (DNA sequence SEQ ID NO:2856; amino acid sequence SEQ IDNO:2857).

Example 10 Test for the Dependence of an Enzyme on the Presence of anUpstream Oligonucleotide

[1256] When choosing a structure-specific nuclease for use in asequential invasive cleavage reaction it is preferable that the enzymehave little ability to cleave a probe 1) in the absence of an upstreamoligonucleotide, and 2) in the absence of overlap between the upstreamoligonucleotide and the downstream probe oligonucleotide. FIGS. 26a-edepicts several structures that can be used to examine the activity ofan enzyme is confronted with each of these types of structures. Thestructure a (FIG. 26a) shows the alignment of a probe oligonucleotidewith a target site on bacteriophage M13 DNA (M13 sequences shown in FIG.26 are provided in SEQ ID NO:217) in the absence of an upstreamoligonucleotide. Structure b (FIG. 26b) is provided with an upstreamoligonucleotide that does not contain a region of overlap with thelabeled probe (the label is indicated by the star). In structures c, dand e (FIGS. 26c-e) the upstream oligonucleotides have overlaps of 1, 3or 5 nucleotides, respectively, with the downstream probeoligonucleotide and each of these structures represents a suitableinvasive cleavage structure. The enzyme Pfu FEN-1 was tested foractivity on each of these structures and all reactions were performed induplicate.

[1257] Each reaction comprised 1 μM 5′ TET labeled probe oligonucleotide89-15-1 (SEQ ID NO:212), 50 nM upstream oligonucleotide (either oligo81-69-2 [SEQ ID NO:216], oligo 81-69-3 [SEQ ID NO:215], oligo 81-69-4[SEQ ID NO:214], oligo 81-69-5 [SEQ ID NO:213], or no upstreamoligonucleotide), 1 fmol M13 target DNA, 10 mg/ml tRNA and 10 ng of PfuFEN-1 in 10 μl of 10 mM MOPS (pH 7.5), 7.5 mM MgCl₂ with 0.05% each ofTween 20 and Nonidet P-40.

[1258] All of the components except the enzyme and the MgCl₂ wereassembled in a final volume of 8 μl and were overlaid with 10 μl ofCHILLOUT liquid wax. The samples were heated to the reaction temperatureof 69° C. The reactions were started by the addition of the Pfu FEN-1and MgCl₂, in a 2 μl volume. After incubation at 69° C. for 30 minutes,the reactions were stopped with 10 μl of 95% formamide, 10 mM EDTA,0.02% methyl violet. Samples were heated to 90° C. for 1 min immediatelybefore electrophoresis through a 20% denaturing acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. Gels were then analyzed with a FMBIO-100 Hitachi FMBIOfluorescence imager. The resulting image is displayed in FIG. 27.

[1259] In FIG. 27, lanes labeled “a” contain the products generated fromreactions conducted without an upstream oligonucleotide (structure a),lanes labeled “b” contain an upstream oligonucleotide that does notinvade the probe/target duplex (structure b). Lanes labeled “c”, “d” and“e” contain the products generated from reactions conducted using anupstream oligonucleotide that invades the probe/target duplex by 1, 3 or5 bases, respectively. The size (in nucleotides) of the uncleaved probeand the cleavage products is indicated to the left of the image in FIG.27. Cleavage of the probe was not detectable when structures a and bwere utilized. In contrast, cleavage products were generated wheninvasive cleavage structures were utilized (structures c-e). These datashow that the Pfu FEN-1 enzyme requires an overlapping upstreamoligonucleotide for specific cleavage of the probe.

[1260] Any enzyme may be examined for its suitability for use in asequential invasive cleavage reaction by examining the ability of thetest enzyme to cleave structures a-e (it is understood by those in theart that the specific oligonucleotide sequences shown in FIGS. 26a-eneed not be employed in the test reactions; these structures are merelyillustrative of suitable test structures). Desirable enzymes displaylittle or no cleavage of structures a and b and display specificcleavage of structures c-e (i.e., they generate cleavage products of thesize expected from the degree of overlap between the twooligonucleotides employed to form the invasive cleavage structure).

Example 11 Use of the Products of a First Invasive Cleavage Reaction toEnable a Second Invasive Cleavage Reaction with a Net Gain inSensitivity

[1261] As discussed in the Description of The Invention above, thedetection sensitivity of the invasive cleavage reaction can be increasedby the performing a second round of invasive cleavage using the productsof the first reaction to complete the cleavage structure in the secondreaction. In this Example, the use of a probe that, when cleaved in afirst invasive cleavage reaction, forms an integrated INVADERoligonucleotide and target molecule for use in a second invasivecleavage reaction, is illustrated.

[1262] A first probe was designed to contain some internalcomplementarity so that when cleaved in a first invasive cleavagereaction the product (“Cut Probe 1”) could form a target strandcomprising an integral INVADER oligonucleotide. A second probe wasprovided in the reaction that would be cleaved at the intended site whenhybridized to the newly formed target/INVADER molecule. To demonstratethe gain in signal due to the performance of sequential invasivecleavages, a standard invasive cleavage assay, as described above, wasperformed in parallel.

[1263] All reactions were performed in duplicate. Each standard (i.e.,non-sequential) invasive cleavage reaction comprised 1 μM 5′fluorescein-labeled probe oligo 073-182 (5′Fl-AGAAAGGAAGGGAAGAAAGCGAA-3′; SEQ ID NO:591), 10 nM upstream oligo81-69-4 (5′-CTTGACGGGGAAAGCCGGCGAACGTGGCGA-3′; SEQ ID NO:214), 10 to 100attomoles of M13 target DNA, 10 mg/ml tRNA and 10 ng of Pfu FEN-1 in 10μl of 10 mM MOPS (pH 7.5), 8 mM MgCl₂ with 0.05% each of Tween 20 andNonidet P-40. All of the components except the enzyme and the MgCl₂ wereassembled in a volume of 7 μl and were overlaid with 10 μl of CHILLOUTliquid wax. The samples were heated to the reaction temperature of 62°C. The reactions were started by the addition of the Pfu FEN-1 andMgCl₂, in a 2 μl volume. After incubation at 62° C. for 30 minutes, thereactions were stopped with 10 μl of 95% formamide, 10 mM EDTA, 0.02%methyl violet.

[1264] Each sequential invasive cleavage reaction comprised 1 μM 5′fluorescein-labeled oligonucleotide 073-191 (the first probe or “Probe1”, 5′ Fl-TGGAGGTCAAAACATCG ATAAGTCGAAGAAAGGAAGGGAAGAAAT-3′; SEQ IDNO:592), 10 nM upstream oligonucleotide 81-69-4 (5′-CTTGACGGGGAAAGCCGGCGAACGTGGCGA-3′; SEQ ID NO:214), 1 μM of 5′ fluorescein labeledoligonucleotide 106-32 (the second probe or “Probe 2”, 5Fl-TGTTTTGACCTCCA-3′; SEQ ID NO:593), 1 to 100 amol of M13 target DNA, 10 mg/ml tRNAand 10 ng of Pfu FEN-1 in 10 μl of 10 mM MOPS (pH 7.5), 8 mM MgCl₂ with0.05% each of Tween 20 and Nonidet P-40. All of the components exceptthe enzyme and the MgCl₂ were assembled in a volume of 8 μl and wereoverlaid with 10 μl of CHILLOUT liquid wax. The samples were heated tothe reaction temperature of 62° C. (this temperature is the optimumtemperature for annealing of Probe 1 to the first target). The reactionswere started by the addition of Pfu FEN-1 and MgCl₂, in a 2 μl volume.After incubation at 62° C. for 15 minutes, the temperature was loweredto 58° C. (this temperature is the optimum temperature for annealing ofProbe 2 to the second target) and the samples were incubated for another15 min. Reactions were stopped by the addition of 10 μl of 95%formamide, 20 mM EDTA, 0.02% methyl violet.

[1265] Samples from both the standard and the sequential invasivecleavage reactions were heated to 90° C. for 1 min immediately beforeelectrophoresis through a 20% denaturing acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. The gel was then analyzed with a Molecular DynamicsFluorImager 595. The resulting image is displayed in FIG. 28a. A graphshowing measure of fluorescence intensity for each of the product bandsis shown in FIG. 28b.

[1266] In FIG. 28a, lanes 1-5 contain the products generated in standardinvasive cleavage reactions that contained either no target (lane 1), 10amol of target (lanes 2 and 3) or with 100 amol of target (lanes 4 and5). The uncleaved probe is seen as a dark band in each lane about halfway down the panel and the cleavage products appear as a smaller blackband near the bottom of the panel, the position of the cleavage productis indicated by an arrow head to the left of FIG. 28a. The gray ladderof bands seen in lanes 1-5 is due to the thermal degradation of theprobe and is not related to the presence or absence of the target DNA.The remaining lanes display products generated in sequential invasivecleavage reactions that contained 1 amol of target (lanes 6 and 7), 10amol of target (lanes 8 and 9) and 100 amol of target (lanes 10 and 11).The uncleaved first probe (Probe 1; labeled “1 uncut”) is seen near thetop of the panel, while the cleaved first probe is indicated as “1:cut”. Similarly, the uncleaved and cleaved second probe are indicated as“2: uncut” and 2: cut,” respectively.

[1267] The graph shown in FIG. 28b compares the amount of productgenerated from the standard reaction (“Series 1”) to the amount ofproduct generated from the second step of the sequential reaction(“Series 2”). The level of background fluorescence measured from areaction that lacked target DNA was subtracted from each measurement. Itcan be seen from the table located below the graph that the signal fromthe standard invasive cleavage assay that contained 100 attomoles oftarget DNA was nearly identical to the signal from the sequentialinvasive cleavage assay in which 1 attomole of target was present,indicating that the inclusion of a second cleavage structure increasesthe sensitivity of the assay 100 to 200-fold. This boost in signalallows easy detection of target nucleic acids at the sub attomole levelusing the sequential invasive cleavage assay, while the standard assay,when performed using this enzyme for only 30 minutes, does not generatedetectable product in the presence of 10 attomoles of target.

[1268] When the amount of target was decreased by 10 or 100 fold in thesequential invasive cleavage assay, the intensity of the signal wasdecreased by the same proportion. This indicates that the quantitativecapability of the invasive cleavage assay is retained even whenreactions are performed in series, thus providing a nucleic aciddetection method that is both sensitive and quantitative.

[1269] While in this Example, the two probes used had different optimalhybridization temperatures (i.e., the temperature empiracally determinedto give the greatest turnover rate in the given reaction conditions),the probes may also be selected (i.e., designed) to have the sameoptimal hybridization temperature so that a temperature shift duringincubation is not necessary.

Example 12 The Products of a Completed Sequential Invasive CleavageReaction Cannot Cross Contaminate Subsequent Similar Reactions

[1270] As discussed in the Description of the Invention, the serialnature of the multiple invasive cleavage events that occur in thesequential invasive cleavage reaction, in contrast to the reciprocatingnature of the polymerase chain reaction and similar doubling assays,means that the sequential invasive cleavage reaction is not subject tocontamination by the products of like reactions because the products ofthe first cleavage reaction do not participate in the generation of newsignal in the second cleavage reaction. If a large amount of a completedreaction were to be added to a newly assembled reaction, the backgroundthat would be produced would come from the amount of target that wasalso carried in, combined with the amount of already-cleaved probe thatwas carried in. In this Example, it is demonstrated that a very largeportion of a primary reaction must be introduced into the secondaryreaction to create significant signal.

[1271] A first or primary sequential invasive cleavage reaction wasperformed as described above using 100 amol of target DNA. A second setof 5 reactions were assembled as described in Ex. 11 with the exceptionthat portions of the first reaction were introduced and no additionaltarget DNA was included. These secondary reactions were initiated andincubated as described above, and included 0, 0.01, 0.1, 1, or 10% ofthe first reaction material. A control reaction including 100 amol oftarget was included in the second set also. The reactions were stopped,resolved by electrophoresis and visualized as described above, and theresulting image is displayed in FIG. 29. The primary probe, uncut secondprobe and the cut 2nd probe are indicated on the left as “1: cut”, 2:uncut” and 2: cut”, respectively.

[1272] In FIG. 29, lane 1 shows the results of the first reaction withthe accumulated product at the bottom of the panel, and lane 2 show a1:10 dilution of the same reaction, to demonstrate the level of signalthat could be expected from that level of contamination, without furtheramplification. Lanes 3 through 7 show the results of the secondarycleavage reactions that contained 0, 10, 1, 0.1 or 0.0 1% of the firstreaction material added as contaminant, respectively and lane 8 shows acontrol reaction that had 100 amol of target DNA added to verify theactivity of the system in the secondary reaction. The signal level inlane 4 is as would be expected when 10% of the pre-cleaved material istransferred (as in lane 2) and 10% of the transferred target materialfrom the lane 1 reaction is allowed to further amplify. At all levels offurther dilution the signal is not readily distinguished frombackground. These data demonstrate that while a large-scale transferfrom one reaction to another may be detectable, cross contamination bythe minute quantities that would be expected from aerosol or fromequipment contamination would not be easily mistaken for a falsepositive result. These data also demonstrate that when the products ofone reaction are deliberately carried over into a fresh sample, theseproducts do not participate in the new reaction, and thus do not affectthe level of target-dependent signal that may be generated in thatreaction.

Example 13 Detection of Human Cytomegalovirus Viral DNA by InvasiveCleavage

[1273] The previous Example demonstrates the ability of the invasivecleavage reaction to detect minute quantities of viral DNA in thepresence of human genomic DNA. In this Example, the probe and INVADERoligonucleotides were designed to target the 3104-3061 region of themajor immediate early gene of human cytomegalovirus (HCMV) as shown inFIG. 30. In FIG. 30, the INVADER oligo (89-44; SEQ ID NO:219) and thefluorescein (Fl)-labeled probe oligo (89-76; SEQ ID NO:218) are shownannealed along a region of the HCMV genome corresponding to nucleotides3057-3110 of the viral DNA (SEQ ID NO:220). The probe used in thisExample is a poly-pyrimidine probe and as shown herein the use of apoly-pyrimidine probe reduces background signal generated by the thermalbreakage of probe oligos.

[1274] The genomic viral DNA was purchased from AdvancedBiotechnologies, Incorporated (Columbia, Md.). The DNA was estimated(but not certified) by personnel at Advanced Biotechnologies to be at aconcentration of 170 amol (1×10⁸ copies) per microliter. The reactionswere performed in quadruplicate. Each reaction comprised 1 μM 5′fluorescein labeled probe oligonucleotide 89-76 (SEQ ID NO:218), 100 nMINVADER oligonucleotide 89-44 (SEQ ID NO:219), 1 ng/ml human genomicDNA, and one of five concentrations of target HCMV DNA in the amountsindicated above each lane in FIG. 31, and 10 ng of Pfu FEN-1 in 10 μl of10 mM MOPS (pH 7.5), 6 mM MgCl₂ with 0.05% each of Tween 20 and NonidetP-40. All of the components except the labeled probe, enzyme and MgCl₂were assembled in a final volume of 7 μl and were overlaid with 10 μl ofCHILLOUT liquid wax. The samples were heated to 95° C. for 5 min, thenreduced to 62° C. The reactions were started by the addition of probe,Pfu FEN-1 and MgCl₂, in a 3 μl volume. After incubation at 62° C. for 60minutes, the reactions were stopped with 10 μl of 95% formamide, 10 mMEDTA, 0.02% methyl violet. Samples were heated to 90° C. for 1 minimmediately before electrophoresis through a 20% acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. Gels were then analyzed with a Molecular DynamicsFluorImager 595.

[1275] The resulting image is displayed in FIG. 31. The replicatereactions were run in groups of four lanes with the target HCMV DNAcontent of the reactions indicated above each set of lanes (0-170 amol).The uncleaved probe is seen in the upper third of the panel (“Uncut89-76”) while the cleavage products are seen in the lower two-thirds ofthe panel (“Cut 89-76”). It can be seen that the intensity of theaccumulated cleavage product is proportional to the amount of the targetDNA in the reaction. Furthermore, it can be clearly seen in reactionsthat did not contain target DNA (“no target”) that the probe is notcleaved, even in a background of human genomic DNA. While 10 ng of humangenomic DNA was included in each of the reactions shown in FIG. 31,inclusion of genomic DNA up to 200 ng has slight impact on the amount ofproduct accumulated. The data did not suggest that 200 ng per 10 μl ofreaction mixture represented the maximum amount of genomic DNA thatcould be tolerated without a significant reduction in signalaccumulation. For reference, this amount of DNA exceeds what might befound in 0.2 ml of urine (a commonly tested amount for HCMV in neonates)and is equivalent to the amount that would be found in about 5 μl ofwhole blood.

[1276] These results demonstrate that the standard (i.e.,non-sequential) invasive cleavage reaction is a sensitive, specific andreproducible means of detecting viral DNA. Detection of 1.7 amol oftarget is roughly equivalent to detection of 10⁶ copies of the virus.This is equivalent to the number of viral genomes that might be found in0.2 mls of urine from a congenitally infected neonate (102 to 106 genomeequivalents per 0.2 mls; Stagno et al., J. Infect. Dis., 132:568[1975]). Use of the sequential invasive cleavage assay would permitdetection of even fewer viral DNA molecules, facilitating detection inblood (10¹ to 10⁵ viral particles per ml; Pector et al., J. Clin.Microbiol., 30:2359 [1992]), which carries a much larger amount ofheterologous DNA.

[1277] From the above it is clear that the invention provides reagentsand methods to permit the detection and characterization of nucleic acidsequences and variations in nucleic acid sequences. The INVADER-directedcleavage reaction and the sequential INVADER-directed cleavage reactionof the present invention provide ideal direct detection methods thatcombine the advantages of the direct detection assays (e.g., easyquantification and minimal risk of carry-over contamination) with thespecificity provided by a dual or tri oligonucleotide hybridizationassay.

[1278] As indicated in the Description of the Invention, the use ofsequential invasive cleavage reactions can present the problem ofresidual uncut first, or primary, probe interacting with the secondarytarget, and either competing with the cut probe for binding, or creatingbackground through low level cleavage of the resulting structure. Thisis shown diagramatically in FIGS. 32 and 33. In FIG. 32, the reactiondepicted makes use of the cleavage product from the first cleavagestructure to form an INVADER oligonucleotide for a second cleavagereaction. The structure formed between the secondary target, thesecondary probe and the uncut primary probe is depicted in FIG. 32, asthe right hand structure shown in step 2a. This structure is recognizedand cleaved by the 5′ nucleases, albeit very inefficiently (i.e., atless than about 1% in most reaction conditions). Nonetheless, theresulting product is indistinguishable from the specific product, andthus may lead to a false positive result. The same effect can occur whenthe cleaved primary probe creates and integrated INVADER/target (IT)molecule, as described in Example 11; the formation of the undesirablecomplex is depicted schematically in FIG. 33, as the right handstructure shown in step 2a.

[1279] The improvements provided by the inclusion of ARRESTORoligonucleotides of various compositions in each of these types ofsequential INVADER assays are demonstrated in the following Examples.These ARRESTOR oligonucleotides are configured to bind the residualuncut probe from the first cleavage reaction in the series, therebyincreasing the efficacy of and reducing the non-specific background inthe subsequent reaction(s).

Example 14 “ARRESTOR” Oligonucleotides Improve Sensitivity of MultipleSequential Invasive Cleavage Assays

[1280] In this Example, the effect of including an ARRESTORoligonucleotide on the generation of signal using the IT probe system 33is demonstrated. The ARRESTOR oligonucleotide hybridizes to the primaryprobe, mainly in the portion that recognizes the target nucleic acidduring the first cleavage reaction. In addition to examining the effectsof adding an ARRESTOR oligonucleotide, the effects of using ARRESTORoligonucleotides that extended in complementarity different distancesinto the region of the primary probe that composes the secondary ITstructure were also investigated. These effects were compared inreactions that included the target DNA over a range of concentrations,or that lacked target DNA, in order to demonstrate the level ofnonspecific (i.e., not related to target nucleic acid) background ineach set of reaction conditions.

[1281] The target DNA for these reactions was a fragment that comprisedthe full length of the hepatitis B genome from strain of serotype adw.This material was created using the polymerase chain reaction fromplasmid pAM6 (ATCC #45020D). The PCRs were conducted using avector-based forward primer, oligo # 156-022-001(5′-ggcgaccacacccgtcctgt-3′; SEQ ID NO:594) and a reverse primer, oligo#156-022-02 (5′-ccacgatgcgtccggcgtag-3′; SEQ ID NO:595) to amplify thefull length of the HBV insert, an amplicon of about 3.2 kb. The cyclingconditions included a denaturation of the plasmid at 95° C. for 5minutes, followed by 30 cycles of 95° C., 30 seconds; 60° C., 40seconds; and 72° C., 4 minutes. This was followed by a final extensionat 72° C. for 10 minutes. The resulting amplicon, termed pAM6#2, wasadjusted to 2 M NH₄OAc, and collected by precipitation wiht isopropanol.After drying in vacuo, DNA was dissolved in 10 mM Tris pH 0.0, 0.1 mMEDTA. The concentration was determined by OD₂₀₀ measurement, and byINVADER assay with comparison to a standard of known concentration.

[1282] The INVADER reactions were conducted as follows. Five mastermixes, termed “A,” “B,” “C,” “D,” and “E,” were assembled; all mixescontained 12.5 mM MOPS, pH 7.5, 500 fmoles primary INVADER oligo#218-55-05 (SEQ ID NO:596), 10 ng human genomic DNA (Novagen) and 30 ngAfuFEN1 enzyme, for every 8 μl of mix. Mix A contained no added HBVgenomic amplicon DNA; mix B contained 600 molecules of HBV genomicamplicon DNA pAM6 #2; mix C contained 6,000 molecules pAM6 #2; mix Dcontained 60,000 molecules pAM6 #2; and mix E contained 600,000molecules pAM6 #2. The mixes were aliquotted to the reaction tubes, 8μl/tube: mix A to tubes 1, 2, 11, 12, 21 and 22; mix B to tubes 3, 4,13, 14, 23 and 24; mix C to tubes 5, 6, 15, 16, 25 and 26; mix D totubes 7, 8, 17, 18, 27 and 28; and mix E to tubes 9, 10, 19, 20, 29 and30. The samples were incubated at 95° C. for 4 minutes to denature theHBV genomic amplicon DNA. The reactions were then cooled to 67° C., and2 μl of a mix containing 37.5 mM MgCl₂ and 2.5 pmoles 218-95-06 (SEQ IDNO:597) for every 2 μl, was added to each sample. The samples wereincubated at 67° C. for 60 minutes. Three secondary reaction mastermixes were prepared, all mixes contained 10 pmoles of secondary probeoligonucleotide #228-48-04 (SEQ ID NO:598) for every 2 μl of mix. Mix 2Acontained no additional oligonucleotide, mix 2B contained 5 pmoles“ARRESTOR” oligo # 218-95-03 (SEQ ID NO:599) and mix 2C contained 5pmoles of “ARRESTOR” oligo # 218-95-01 (SEQ ID NO:600). After the 60minute incubation at 67° C. (the primary reaction described above), 2 μlof the secondary reaction mix was added to each sample: Mix 2A was addedto samples #1-10; Mix 2B was added to samples #11-20; and Mix 2C wasadded to samples #21-30. The temperature was adjusted to 52° C. and thesamples were incubated for 30 minutes at 52° C. The reactions were thenstopped by the addition of 10 μl of a solution of 95% formamide, 5 mMEDTA and 0.02% crystal violet. All samples were heated to 95° C. for 2minutes, and 4 μl of each sample were resolved by electrophoresisthrough 20% denaturing acrylamide gel (19:1 cross-linked) with 7 M urea,in a buffer containing 45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. Theresults were imaged using the Molecular Dynamics Fluoroimager 595,excitation 488, emission 530. The resulting images are shown in FIG. 34.

[1283] In FIG. 34, Panel A shows the results of the target titrationwhen no ARRESTOR oligonucleotide was included in the secondary reaction;Panel B shows the results of the same target titration using an ARRESTORoligonucleotide that extended 2 nt into the non-target complementaryregion of the primary probe; and Panel C shows the results of the sametarget titration using an ARRESTOR oligonucleotide that extended 4 ntinto the non-target complementary region of the primary probe. Theproduct of the secondary cleavage reaction is seen as a band near thebottom of each panel. The first two lanes of each panel (i.e., 1 and 2,11 and 12, 21 and 22) lacked target DNA, and the signal that co-migrateswith the product band represents the nonspecific background under eachset of conditions.

[1284] It can be seen by visual inspection of these panels that thebackground signal is both reduced, and made more predictable, by theinclusion of either species of ARRESTOR oligonucleotide. In addition toreducing the background in the no-target control lanes, the backgroundreduction in the reactions that had the more dilute amounts of targetincluded is reduced, leading to a signal that is a more accuratereflection of the target contained within the reaction, thus improvingthe quantitative range of the multiple, sequential invasive cleavagereaction.

[1285] To quantify the impact of including the ARRESTOR oligonucleotidein the secondary cleavage reaction under these conditions, the averageproduct band signal from the reactions having the largest amount oftarget (i.e., averages of the signals from lanes 9 and 10, lanes 19 and20, and lanes 29 and 30), were compared to the averaged signal from theno-target contol lanes for each panel, determine the “fold overbackground,” the factor of signal amplification over background, undereach set of conditions. For the reactions without the ARRESTORoligonucleotide, Panel A, the fold over background was 5.3; for Panel B,the fold over background was 12.7; and for Panel C, the fold overbackground was 13.4, indicating that in this system inclusion of anyARRESTOR oligonucleotide at least doubled the specificity of the signalover the ARRESTOR oligonucleotide-less reactions, and that the ARRESTORoligonucleotide that extended slightly farther into the non-targetcomplementary region may be slightly more effective, at least in thisembodiment of the system. This clearly shows the benefits of using anARRESTOR oligonucleotide to enhance the specificity of these reactions,an advantage that is of particular benefit at low levels of targetnucleic acid.

Example 15 “ARRESTOR” Oligonucleotides Allow use of HigherConcentrations of Primary Probe Without Increasing Background Signal

[1286] Increasing the concentration of the probe in the invasivecleavage reaction can dramatically increase the amount of signalgenerated for a given amount of target DNA. While not intending to limitthe explanation to any specific mechanism, this is believed to be causedby the fact that increased concentration of probe increases the rate atwhich the cleaved probe is supplanted by an uncleaved copy, therebyincreasing the apparent turnover rate of the cleavage reaction.Unfortunately, this effect could not heretofore be applied in theprimary cleavage reaction of a multiple sequential INVADER assay becausethe residual uncleaved primary probe can hybridize to the secondarytarget, in competition with the cleaved molecules, thereby reducing theefficacy of the secondary reaction. Elevated concentrations of primaryprobe exacerbate this problem. Further, the resulting complexes, asdescribed above, can be cleaved at a low level, contributing tobackground. Therefore, increasing the primary probe can have the doublenegative effect of both slowing the secondary reaction and increasingthe level of this form of non target-specific background. The use of anARRESTOR oligonucleotide to sequester or neutralize the residual primaryprobe allows this concentration-enhancing effect to be applied to thesesequential reactions.

[1287] To demonstrate this effect, two sets of reactions were conducted.In the first set of reactions, the reactions were conducted using arange of primary probe concentrations, but no ARRESTOR oligonucleotidewas supplied in the secondary reaction. In the second set of reactions,the same probe concentrations were used, but an ARRESTOR oligonucleotidewas added for the secondary reactions.

[1288] All reactions were performed in duplicate. Primary INVADERreactions were done in a final volume of 10 μl and contained: 10 mMMOPS, pH 7.5, 7.5 mM MgCl₂, 500 fin of primary INVADER (218-55-05; SEQID NO:596); 30 ng of AfuFEN1 enzyme and 10 ng of human genomic DNA. 100zeptomoles of HBV pAM6 #2 amplicon was included in all even numberedreactions (by reference to FIGS. 35A and B). Reactions included 10pmoles, 20 pmoles, 50 pmoles, 100 pmoles or 150 pmoles of primary probe(218-55-02; SEQ ID NO:601). MOPS, target and INVADER oligonucleotideswere combined to a final volume of 7 μl. Samples were heat denatured at95° C. for 5 minutes, then cooled to 67° C. During the 5 minutedenaturation, MgCl₂, probe and enzyme were combined. The primary INVADERreactions were initiated by the addition of 3 μl of MgCl₂, probe andenzyme mix, to the final concentrations indicated above. Reactions wereincubated for 30 minutes at 67° C. The reactions were then cooled to 52°C., and each primary INVADER reaction received the following secondaryreaction components in a total volume of 4 μl: 2.5 pmoles secondarytarget (oligo number 218-95-04; SEQ ID NO:602); 10 pmoles secondaryprobe (oligo number 228-48-04; SEQ ID NO:598). The reactions thatincluded the ARRESTOR oligonucleotide had either 40 pmoles, 80 pmoles,200 pmoles, 400 pmoles or 600 pmoles of ARRESTOR oligonucleotide (oligonumber 218-95-01; SEQ ID NO:600), added at a 4-fold molar excess overthe primary probe amount for each reaction, with this mix. Reactionswere then incubated at 52° C. for 30 minutes. The reactions were stoppedby the addition of 10 μl of a solution of 95% formamide, 10 mM EDTA and0.02% crystal violet. All samples were heated to 95° C. for 1 minute,and 4 μl of each sample were resolved by electrophoresis through 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. The results wereimaged using the Molecular Dynamics Fluoroimager 595, excitation 488,emission 530. The resulting images for the reactions either without orwith an ARRESTOR oligonucleotide are shown in FIGS. 35A and 35B,respectively. The products of cleavage of the secondary probe are seenas a band near the bottom of each panel.

[1289] In FIG. 35A, lane sets 1 and 2 show results with 10 pmoles ofprimary probe; 3 and 4 had 20 pmoles; 5 and 6 had 50 pmoles; 7 and 8 had100 pmoles; and 9 and 10 had 150 pmoles. It can be seen by visualexamination, that the increases in the amount of primary probe have thecombined effect of slightly increasing the background in the no-targetlanes (odd numbers) while reducing the specific signal in the presenceof target (even numbered lanes), and therefore the reducing thespecificity of the reaction if viewed as the measure of “fold overbackground,” demonstrating that the approach of increasing signal byincreasing probe cannot be applied in these sequential reactions.

[1290] In FIG. 35B, lane sets 1 and 2 show results with 10 pmoles ofprimary probe; while 3 and 4 had 20 pmoles; 5 and 6 had 50 pmoles; 7 and8 had 100 pmoles; and 9 and 10 had 150 pmoles. In addition, eachreaction included 4-fold molar excess of the ARRESTOR oligonucleotideadded before the secondary cleavage reaction. It can be seen by visualexamination that the background in the no-target lanes (odd numbers) islower in all cases, while the specific signal in the presence of target(even numbered lanes) increases with increased amounts of primary probe,leading to a greater “fold over background” sensitivity at this targetlevel.

[1291] To quantitatively compare these effects, the fluorescence signalfrom the products of both non-specific and specific cleavage weremeasured. The results are depicted graphically in FIG. 35C, graphed as ameasure of the percentage of the secondary probe cleaved during thereaction, compared to the amount of primary probe used. Examination ofthe plots from the no-target reactions confirms that the background inthe absence of the ARRESTOR oligonucleotide is, in general, roughlytwo-fold higher, and that both increase slightly with the increasingprobe amounts. The specific signals however, diverge between the twosets of reaction more dramatically. While the signal in the no-ARRESTORoligonucleotide reactions decreases steadily as primary probe wasincreased, the signal in the ARRESTOR oligonucleotide reactionscontinued to increase. At the highest primary probe concentrationstested, the no-ARRESTOR oligonucleotide reactions had specific signalthat was only 1.7 fold over background, while the ARRESTORoligonucleotide reactions detected the 100 zmoles (60,000 copies) oftarget with a signal 6.5 fold over background, thus demonstrating theimprovement in the sequential invasive cleavage reaction when anARRESTOR oligonucleotide is included.

Example 16 Modified Backbones Improve Performance of ARRESTOROligonucleotides All Natural “ARRESTOR” Oligo with No 3′-Amine

[1292] The reactions described in the previous two Examples usedARRESTOR oligonucleotides that were constructed using 2′ O-methyl ribosebackbone, and that included a positively charged amine group on the 3′terminal nucleotide. The modifications were made specifically to reduceenzyme interaction with the primary probe/ARRESTOR oligonucleotidecomplex. During the development of the present invention, it wasdetermined that the 2′ O-methy modified oligonucleotides are somewhatresistant to cleavage by the 5′ nucleases, just as they are slowlydegraded by nucleases when used in antisense applications (See e.g.,Kawasaki et al., J. Med. Chem., 36:831 [1993]).

[1293] The presence of an amino group on the 3′ end of anoligonucleotide reduces its ability to direct invasive cleavage. Toreduce the possibility that the ARRESTOR oligonucleotide would form acleavage structure in this way, an amino group was included in thedesign of the experiments described in this and other Examples.

[1294] Initial designs of the ARRESTOR oligonucleotides (sometimesreferred to as “blockers”) did not include these modifications, andthese molecules were found to provide no benefit in reducing backgroundcleavage in the sequential invasive cleavage assay and, in fact,sometimes contributed to background by inducing cleavage at anunanticipated site, presumably by providing some element to analternative cleavage structure. The effects of natural and modifiedARRESTOR oligonucleotides on the background noise in these reactions areexamined in this Example.

[1295] The efficacy of an “all-natural ARRESTOR oligonucleotide (i.e.,an ARRESTOR oligonucleotide that did not contain any base analogs ormodifications) was examined by comparison to an identical reactions thatlacked ARRESTOR oligonucleotide. All reactions were performed induplicate, and were conducted as follows. Two master mixes wereassembled, each containing 12.5 mM MOPS, pH 7.5, 500 fmoles primaryINVADER oligonucleotide #218-55-05 (SEQ ID NO:596), 10 ng human genomicDNA (Novagen) and 30 ng AfuFEN1 enzyme for every 8 μl of mix. Mix Acontained no added HBV genomic amplicon DNA, mix B contained 600,000molecules of HBV genomic amplicon DNA, pAM6 #2. The mixes weredistributed to the reaction tubes, in aliquots of 8 μl/tube as follows:mix A to tubes 1, 2, 5 and 6; and mix B to tubes 3, 4, 7 and 8. Thesamples were incubated at 95° C. for 4 minutes to denature the HBVgenomic amplicon DNA. The reactions were then cooled to 67° C. and 2 ulof a mix containing 37.5 mM MgCl₂ and 10 pmoles 218-55-02B (SEQ IDNO:603) for every 2 μl, was added to each sample. The samples were thenincubated at 67° C. for 30 minutes. Two secondary reaction master mixeswere prepared, each containing 10 pmoles of secondary probe oligo#228-48-04N (SEQ ID NO:604) and 2.5 pmoles of secondary targetoligonucleotide #218-95-04 (SEQ ID NO:602) for every 3 μl of mix. Mix 2Acontained no additional oligonucleotide, while mix 2B contained 50pmoles of the natural “ARRESTOR” oligonucleotide #241-62-02 (SEQ IDNO:605). After the initial 30 minute incubation at 67° C., thetemperature was adjusted to 52° C., and 3 μl of a secondary reaction mixwas added to each sample, as follows: Mix 2A was added to samples #1-4;and Mix 2B was added to samples #5-8. The samples were then incubatedfor 30 minutes at 52° C. The reactions were then stopped by the additionof 10 μl of a solution of 95% formamide, 10 mM EDTA and 0.02% crystalviolet.

[1296] All of the samples were heated to 95° C. for 2 minutes, and 4 μlof each sample were resolved by electrophoresis through a 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. The results were imagedusing the Molecular Dynamics Fluoroimager 595, excitation 488, emission530. The resulting image is shown in FIG. 36A.

[1297] To compare the effects of the various modifications made to theARRESTOR oligonucleotides, reactions were performed using ARRESTORoligonucleotides having all natural bases, but including a 3′ terminalamine; ARRESTOR oligonucleotides having a 3′ portion composed of 2′O-methyl nucleotides, plus the 3′ terminal amine; and ARRESTORoligonucleotides composed entirely of 2′ O-methyl nucleotides, plus the3′ terminal amine. These were compared to reactions performed without anARRESTOR oligonucleotide. The reactions were conducted as follows. Twomaster mixes were assembled, all mixes contained 14.3 mM MOPS, pH 7.5,500 fmoles primary INVADER oligo #218-55-05 (SEQ ID NO:596) and 10 nghuman genomic DNA (Novagen) for every 7 μl of mix. Mix A contained noadded HBV genomic amplicon DNA, mix B contained 600,000 molecules of HBVgenomic amplicon DNA, pAM6 #2. The mixes were distributed to thereaction tubes, at 7 μl/tube: mix A to tubes 1, 2, 5, 6, 9, 10, 13 and14; and mix B to tubes 3, 4, 7, 8, 11, 12, 15 and 16. The samples werewarmed to 95° C. for 4 minutes to denature the HBV DNA. The reactionswere then cooled to 67° C. and 3 l of a mix containing 25 mM MgCl₂, 25pmoles 218-55-02B (SEQ ID NO:603) and 30 ng AfuFEN1 enzyme per 3 μl,were added to each sample. The samples were then incubated at 67° C. for30 minutes. Four secondary reaction master mixes were prepared; allmixes contained 10 pmoles of secondary probe oligonucleotide #228-48-04B(SEQ ID NO:606) and 2.5 pmoles of secondary target oligonucleotide#218-95-04 (SEQ ID NO:602) for every 3 μl of mix. Mix 2A contained noadditional oligonucleotide, while mix 2B contained 100 pmoles of thenatural+amine ARRESTOR oligonucleotide # 241-62-01 (SEQ ID NO:607), mix2C contained 100 pmoles of partially O-methyl+amine oligonucleotide #241-62-03 (SEQ ID NO:608) and mix 2D contained 100 pmoles of allO-methyl+amine oligonucleotide # 241-64-01 (SEQ ID NO:609). After theinitial 30 minute incubation at 67° C., the temperature was adjusted to52° C. and 3 μl of a secondary reaction mix was added to each sample, asfollows: mix 2A was added to samples #1-4; mix 2B was added to samples#5-8; mix 2C was added to samples #9-12; and mix 2D was added to samples#13-16. The samples were incubated for 30 minutes at 52° C., thenstopped by the addition of 10 μl of a solution of 95% formamide, 10 mMNaEDTA, and 0.2% crystal violet.

[1298] All samples were heated to 95° C. for 2 minutes, and 4 μl of eachsample were resolved by electrophoresis through a 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. The results were imagedusing the Molecular Dynamics Fluoroimager 595, excitation 488, emission530. The resulting image is shown in FIG. 36B.

[1299] In FIG. 36A, the left-hand panel shows the reactions that lackedan ARRESTOR oligonucleotide, while the right hand panel shows the datafrom reactions that included the all natural ARRESTOR oligonucleotide.The first two lanes of each panel are from no-target controls, thesecond set of lanes contained target. The products of cleavage arevisible in the bottom one/fourth of each panel. The position at whichthe specific reaction products should run is indicated by arrows on leftand right.

[1300] It can be seen by examination of these data, that the reactionsrun in the absence of ARRESTOR oligonucleotide show reproducible qualitybetween the replicates, and show significant cleavage only when targetis present. In contrast, the addition of another unmodifiedoligonucleotide into the reactions causes great variation between thereplicate lanes (e.g., lanes 5 and 6 were provided with the samereactants, but produced markedly different results). The introduction ofthe all-natural ARRESTOR oligonucleotide produced, rather than reduced,background in these no-target lanes, and increased cleavage at othersites (i.e., the bands other that those indicated by the arrows flankingthe panels). For these reasons the modifications that are describedabove, the effects of which are shown on FIG. 36B, were incorporated.

[1301] The first 4 lanes of FIG. 36B show the products of duplicatereactions without an ARRESTOR oligonucleotide, plus or minus the HBVtarget (lanes 1, 2, and lanes 3, 4, respectively); The next 4 lanes, 5,6 and 7, 8 used a natural ARRESTOR oligonucleotide having a 3′ terminalamine; lanes 9, 10 and 11, 12 used the ARRESTOR oligonucleotide with a3′ portion composed of 2′ O-methyl nucleotides, and having a 3′ terminalamine; lanes 13, 14 and 15, 16 used the ARRESTOR oligonucleotidecomposed entirely of 2′ O-methyl nucleotides and having a 3′ terminalamine. The products of cleavage of the secondary probe are visible inthe lower one third of each panel.

[1302] Visual inspection of these data shows that the addition of the 3′terminal amine to the natural ARRESTOR oligonucleotide suppresses theaberrant cleavage seen in FIG. 36A, but this ARRESTOR oligonucleotidedoes not improve the performance of the reaction, as compared to theno-ARRESTOR oligonucleotide controls. In contrast, the use of the 2′O-methyl nucleotides in the body of the ARRESTOR oligonucleotide doesreduce background, whether partially or completely substituted. Toquantify the relative effects of these modifications, the fluorescencefrom each of the co-migrating product bands was measured, the signalsfrom the duplicate lanes were averaged and the “fold over background”was calculated for each reaction containing target nucleic acid.

[1303] When ARRESTOR oligonucleotide was omitted, the target-specificsignal (lanes 3, 4) was 27-fold over the no target background; thenatural ARRESTOR oligonucleotide+amine gave a signal of 17-fold overbackground; the partial 2′ O-methyl+amine gave a signal of 47-fold overbackground; and the completely 2′ O-methyl+amine gave a signal of 33fold over background.

[1304] These Figures show that both modifications can have a beneficialeffect on the specificity of the multiple, sequential invasive cleavageassay. They also show that the use of the 2′ O-methyl substitutedbackbone, either partial or entire, markedly improves the specificity ofthese reactions. It is intended that, in various embodiments of thepresent inventon, any number of modifications that make either theARRESTOR oligonucleotide or the complex it forms with the primary targetresistant to nucleases will provide similar enhancement.

Example 17 Effect of ARRESTOR Oligonucleotide Length on SignalEnhancement in Multiple Sequential Invasive Cleavage Assays

[1305] As noted in the Description of the Invention, the optimal lengthfor an ARRESTOR oligonucleotide depends upon the design of the othernucleic acid elements of the INVADER reaction, particularly on thedesign of the primary probe. In this Example, the effects of varying thelength of the ARRESTOR oligonucleotide were explored in systems usingtwo different secondary probes. A schematic diagram showing theseARRESTOR oligonucleotides aligned as they would hybridize to the primaryprobe oligonucleotide is provided in FIG. 37C. In this Figure, theregion of the primary probe that recognizes the target nucleic acid isshown underlined; the non-underlined portion, plus the first underlinedbase is the portion that is released by the first cleavage, and goes onto participate in the second or subsequent cleavage structure.

[1306] All reactions were performed in duplicate. The INVADER reactionswere done in a final volume of 10 μl final volume containing 10 mM MOPS,pH 7.5, mM MgCl₂, 500 fmoles of primary INVADER 241-95-01, (SEQ IDNO:610), 25 pmoles of primary probe 241-95-02 (SEQ ID NO:611), 30 ng ofAfuFEN1 enzyme, and 10 ng of human genomic DNA, and if included, 1amoles of HBV amplicon pAM 6 #2. MOPS, target DNA, and INVADERoligonucleotides were combined to a final volume of 7 μl. Samples wereheat denatured at 95° C. for 5 minutes, then cooled to 67° C. During the5 minute denaturation, MgCl₂, probe and enzyme were combined. Theprimary INVADER reactions were initiated by the addition of 3 μl ofMgCl₂, probe and enzyme mix, to the final concentrations indicatedabove. Reactions were incubated for 30 minutes at 67° C. The reactionwere then cooled to 52° C., and each primary INVADER reaction receivedthe following secondary reaction components in a total volume of 3 μl:2.5 pmoles secondary target 241-95-07 (SEQ ID NO:612), 10 pmoles ofeither secondary probe 228-48-04 (SEQ ID NO:598), or 228-48-04N (SEQ IDNO:604) and 100 pmoles of an ARRESTOR oligonucleotide, either 241-95-03(SEQ ID NO:613), 241-95-04 (SEQ ID NO:614), 241-95-05 (SEQ ID NO:615) or241-95-06 (SEQ ID NO:616). The ARRESTOR oligonucleotides were omittedfrom some reactions as controls for ARRESTOR oligonucleotide effects.

[1307] The reactions were incubated at 52° C. for 34 minutes, and werethen stopped by the addition of 10 μl of 95% formamide, 10 mM EDTA, and0.02% crystal violet. All samples were heated to 95° C. for 1 minute,and 4 μl of each sample were resolved by electrophoresis through 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. The results wereimaged using the Molecular Dynamics Fluoroimager 595, excitation 488,emission 530. The resulting images for the reactions with the shorterand longer secondary probes are shown in FIGS. 37A and 37B,respectively.

[1308] In each Figure, the products of cleavage are visible as bands inthe bottom half of each lane. The first 4 lanes of each Figure show theproducts of duplicate reactions without an ARRESTOR oligonucleotide,plus or minus the HBV target (lanes sets 1 and 2 respectively); in thenext 4 lanes, sets 3 and 4 used the shortest ARRESTOR 241-95-03 (SEQ IDNO:613); lanes 5 and 6 used 241-95-04 (SEQ ID NO:614); lanes 7 and 8used 241-95-05 (SEQ ID NO:615); and lanes 9 and 10 used 241-95-06 (SEQID NO:616).

[1309] The principal background of concern is the band that appears inthe “no target” control lanes (odd numbers; this band co-migrates withthe target-specific signal near the bottom of each gel panel). Visualinspection shows that the shortest ARRESTOR oligonucleotide was theleast effective at suppressing this background, and that the efficacywas increased when the ARRESTOR oligonucleotide extended further intothe portion that participates in the subsequent cleavage reaction. Evenwith this difference in effect, it can be seen from these data thatthere is much latitude in the design of the ARRESTOR oligonucleotide.The choice of lengths will be influenced by the temperature at which thereaction making use of the ARRESTOR oligonucleotide is performed, thelengths of the duplexes formed between the primary probe and the target,the primary probe and the secondary target, and the relativeconcentrations of the different nucleic acid species in the reactions.

Example 18 Effect of ARRESTOR Oligonucleotide Concentration on SignalEnhancement in Multiple Sequential Invasive Cleavage Assays

[1310] In examining the effects of including ARRESTOR oligonucleotidesin these cleavage reactions, it was of interest to determine if theconcentration of the ARRESTOR oligonucleotide in excess of the primaryprobe concentration would have an effect on yields of eithernon-specific or specific signal, and if the length of the ARRESTORoligonucleotide would be a factor. These two variables were investigatedin the following Example.

[1311] All reactions were performed in duplicate. The primary INVADERreactions were done in a final volume of 10 μl and contained 10 mM MOPS,pH 7.5; 7.5 mM MgCl₂, 500 fmoles of primary INVADER 241-95-01 (SEQ IDNO:610), 25 pmoles of primary probe 241-95-02 (SEQ ID NO:611), 30 ng ofAfuFEN1 enzyme, and 10 ng of human genomic DNA. Where included, thetarget DNA was 1 amole of HBV amplicon pAM 6 #2, as described above.MOPS, target and INVADER were combined to a final volume of 7 μl. Thesamples were heat denatured at 95° C. for 5 minutes, then cooled to 67°C. During the 5 minute denaturation, MgCl₂, probe and enzyme werecombined. The primary INVADER reactions were initiated by the additionof 3 μl of MgCl₂, probe and enzyme mix. The reactions were incubated for30 minutes at 67° C. The reactions were then cooled to 52° C. and eachprimary INVADER reaction received the following secondary reactioncomponents: 2.5 pmoles secondary target 241-95-07 (SEQ ID NO:612), 10pmoles secondary probe 228-48-04 (SEQ ID NO:598); and, if included, 50,100 or 200 pmoles of either ARRESTOR oligonucleotide 241-95-03 (SEQ IDNO:613) or 241-95-05 (SEQ ID NO:615), in a total volume of 3 μl.Reactions were then incubated at 52° C. for 35 minutes. Reactions werestopped by the addition of 10 μl of 95% formamide, 10 mM EDTA, and 0.02%crystal violet. All of the samples were heated to 95° C. for 1 minute,and 4 μl of each sample were resolved by electrophoresis through 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH 8.3), and 1.4 mM EDTA. The results wereimaged using the Molecular Dynamics Fluoroimager 595, excitation 488,emission 530. The resulting images are shown as a composite image inFIG. 38.

[1312] Each of the duplicate reactions were loaded on the gel inadjacent lanes and are labeled with a single lane number. All oddnumbered lanes were no-target controls. Lanes 1 and 2 had no ARRESTORoligonucleotide added; lanes 3-8 show results from reactions containingthe shorter ARRESTOR oligonucleotide, 241-95-03 (SEQ ID NO:613); lanes9-14 show results from reactions containing the longer ARRESTORoligonucleotide, 241-95-05 (SEQ ID NO:615). The products of cleavagefrom the secondary reaction are visible in the bottom one third of eachpanel. Visual inspection of these data (i.e., comparison of the specificproducts to the background bands) shows that both ARRESTORoligonucleotides have some beneficial effect at all concentration.

[1313] To quantify the relative effects of ARRESTOR oligonucleotidelength and concentration, the fluorescence from each of the co-migratingproduct bands was measured, the signals from the duplicate lanes wereaveraged and the “fold over background” (signal+target/signal-target)was calculated for each reaction containing target nucleic acid. Thereaction lacking an ARRESTOR oligonucleotide yielded a signalapproximately 27-fold over background. Inclusion of the shorter ARRESTORoligonucleotide at 50, 100 or 200 pmoles produced products at 42, 51 and60-fold over background, respectively. This shows that while the shortARRESTOR at the lowest concentration seems to be less effective than thelonger ARRESTOR oligonucleotides (See, e.g., previous Example) this canbe compensated for by increasing the concentration of ARRESTORoligonucleotide, and thereby the ARRESTOR oligonucleotide:primary proberatio.

[1314] In contrast, inclusion of the longer ARRESTOR oligonucleotide at50, 100 or 200 pmoles produced products at 60, 32 and 24 fold overbackground, respectively. At the lowest concentration, the efficacy ofthis longer ARRESTOR oligonucleotide relative to the shorter ARRESTORoligonucleotide is consistent with the previous Example. Increasing theconcentration, however, decreased the yield of specific product,suggesting a competition effect with some element of the secondarycleavage reaction.

[1315] These data show that the ARRESTOR oligonucleotides can be used toadvantage in a number of specific reaction designs. The choice ofconcentration will be influenced by the temperature at which thereaction making use of the ARRESTOR oligonucleotide is performed, thelengths of the duplexes formed between the primary probe and the target,the primary probe and the secondary target, and between the primaryprobe and the ARRESTOR oligonucleotide.

[1316] Selection of oligonucleotides for target nucleic acids other thanthe HBV shown here, (e.g., oligonucleotide composition and length), andthe optimization of cleavage reaction conditions in accord with themodels provided here follow routine methods and common practice wellknown to those skilled in the methods of molecular biology.

[1317] Example 10 demonstrated that some enzymes require an overlapbetween an upstream INVADER oligonucleotide and a downstream probeoligonucleotide to create a cleavage structure (FIG. 27). It has alsobeen determined that the 3′ terminal nucleotide of the INVADERoligonucleotide need not be complementary to the target strand, even ifit is the only overlapping base in the INVADER oligonucleotide (e.g., aswith the HCMV probes shown in FIG. 30). The requirement for an overlapcan serve as a convenient basis for detecting single base polymorphisms(SNPs) or mutations in a nucleic acid sample.

[1318] For detection of single base variations, at least twooligonucleotides (e.g., a probe and an INVADER oligonucleotide)hybridize in tandem to the target nucleic acid to form the overlappingstructure recognized by the CLEAVASE enzyme to be used in the reaction.An unpaired “flap” is included on the 5′ end of the probe. The enzymerecognizes the overlap and cleaves off the unpaired flap, releasing itas a target-specific product. Enzymes that have a strong preference foran overlapping structure, i.e., that cleave the overlapping structure ata much greater rate than they cleave a non-overlapping structure includethe FEN-1 enzymes from Archaeoglobus fulgidus and Pyrococcus furiosusand such enzymes are particularly preferred in the detection ofmutations and SNPs.

Example 19 Kits for Performing the mRNA INVADER Assay

[1319] In some embodiments, the present invention provides kitscomprising one or more of the components necessary for practicing thepresent invention. For example, the present invention provides kits forstoring or delivering the enzymes of the present invention and/or thereaction components necessary to practice a cleavage assay (e.g., theINVADER assay). By way of example, and not intending to limit the kitsof the present invention to any particular configuration or combinationof components, the following section describes one embodiment of a kitfor practicing the present invention:

[1320] In some embodiments, the kits of the present invention providethe following reagents: CLEAVASE enzyme (e.g., Primary Oligos TthAKK)RNA Primary Buffer 1 Secondary Oligos RNA Secondary Buffer 1 RNAStandard [100 amol/μl] tRNA Carrier [20 ng/μl] 10X Cell Lysis Buffer 1T₁₀e_(0.1) Buffer [10 mM Tris · HCl, pH 8, 0.1 mM EDTA]

[1321] Examples of Primary Oligonucleotides and SecondaryOligonucleotides suitable for use with the methods of the presentinvention are provided in FIG. 41. While the oligonucleotides showntherein may find use in a number of the methods, and variations of themethods, of the present invention, these INVADER assay oligonucleotidesets find particular use with kits of the present invention. Theoligonucleotide sets shown in FIG. 41 may be used as individual sets todetect individual target RNAs, or may be comined in biplex or multiplexreactions for the detection of two or more analytes or controls in asingle reaction. It is contemplated that the designs of these probessets (e.g., the oligonucleotides and/or their sequences) may be adaptedfor use in DNA detection assays, using the guidelines for reactiondesign and optimization provided herein. Additional oligonucleotidesthat find use in detection assays and kits of the present invention areprovided in FIG. 47.

[1322] In some embodiments, a kit of the present invention provides alist of additional components (e.g., reagents, supplies, and/orequipment) to be supplied by a user in order to perform the methods ofthe invention. For example, and without intending to limit suchadditional components lists to any particular components, one embodimentof such a list comprises the following:

[1323] RNase-free (e.g., DEPC-treated) H₂O

[1324] Clear CHILLOUT-14 liquid wax (MJ Research) or RNase-free, opticalgrade mineral oil (Sigma, Cat. No. M-5904)

[1325] Phosphate-buffered saline (no MgCl₂, no CaCl₂)

[1326] 96-well polypropylene microplate (MJ Research, Cat. No. MSP-9601)

[1327] 0.2-ml thin-wall tubes

[1328] Thermaseal well tape (e.g., GeneMate, Cat. No. T-2417-5)

[1329] Multichannel pipets (0.5-10 μl, 2.5-20 μl, 20-200 μl)

[1330] Thermal cycler or other heat source (e.g., lab oven or heatingblock).

[1331] Fluorescence microplate reader (a preferred plate reader istop-reading, equipped with light filters have the followingcharacteristics: Excitation Emission (Wavelength/Bandwidth)(Wavelength/Bandwidth) 485 nm/20 nm 530 nm/25 nm 560 nm/20 nm 620 nm/40nm

[1332] In some embodiments, a kit of the present invention provides alist of optional components (e.g., reagents, supplies, and/or equipment)to be supplied by a user to facilitate performance of the methods of theinvention. For example, and without intending to limit such optionalcomponents lists to any particular components, one embodiment of such alist comprises the following:

[1333] tRNA Solution, 20 ng/μl (Sigma, R-5636)

[1334] 1×Stop Solution (10 mM Tris.HCl, pH 8, 10 mM EDTA) Black opaque,96-well microplate (e.g., COSTAR, Cat. No. 3915)

[1335] Electronic repeat pipet (250 μl)

[1336] In some embodiments of a kit, detailed protocols are provided. Inpreferred embodiments, protocols for the assembly of INVADER assayreactions (e.g., formulations and preferred procedures for makingreaction mixtures) are provided. In particularly preferred embodiments,protocols for assembly of reaction mixtures include computational orgraphical aids to reduce risk of error in the performance of the methodsof the present invention (e.g., tables to facilitate calculation ofvolumes of reagents needed for multiple reactions, and plate-layoutguides to assist in configuring multi-well assay plates to containnumerous assay reactions). By way of example, and without intending tolimit such protocols to any particular content or format, kits of thepresent invention may comprise the following protocol:

[1337] I. Detailed mRNA Invader Assay Protocol

[1338] 1. Plan the microplate layout for each experimental run. Anexample microplate layout for 40 samples, 6 standards, and a No TargetControl is shown in FIG. 40. Inclusion of a No Target Control (tRNACarrier or 1×Cell Lysis Buffer 1) and quantitation standards arerequired for absolute quantitation.

[1339] 2. Prepare the Primary Reaction Mix for either the single orbiplex assay format. To calculate the volumes of reaction componentsneeded for the assay (X Volume), multiply the number of reactions (forboth samples and controls) by 1.25 [X Volume (μl)=# reactions×1.25].Vortex the Primary Reaction Mix briefly after the last reagent additionto mix thoroughly. Aliquot 5 μl of the Primary Reaction Mix permicroplate well (an electronic repeat pipet is recommended for thisstep). Primary Reaction Mix Reaction Components 1× Volume _× VolumeSingle Assay Format RNA Primary Buffer 1  4.0 μl _Primary Oligos 0.25 μlT₁₀e_(0.1) Buffer 0.25 μl CLEAVASE enz. enzyme  0.5 μl Total Mix Volume(1×)  5.0 μl Biplex Assay Format RNA Primary Buffer 1  4.0 μl _PrimaryOligos 0.25 μl _Housekeeping Primary Oligos 0.25 μl CLEAVASE enzyme  0.5μl Total Mix Volume (1×)  5.0 μl

[1340] 3. Add 5 μl of each No Target Control, standard, or sample (totalRNA or cell lysate) to the appropriate well and mix by pipetting up anddown 1-2 times. Overlay each reaction with 10 μl of clear CHILLOUT ormineral oil. Seal microplate with Thermaseal well tape.

[1341] 4. Incubate reactions for 90 minutes at 60° C. in a thermalcycler or oven.

[1342] 5. While the primary reaction is incubating, prepare theSecondary FRET Reaction Mix for the single or biplex format. Calculatethe component volumes required (X Volume) by multiplying the number ofreactions (for both samples and controls) by 1.25 [X Volume (μl)=#reactions×1.25 (μl)]. Aliquot the Secondary FRET Reaction Mix intomultiple 0.2-ml thin-wall tubes or an 8-well strip (70 μl/tube issufficient for a row of 12 reactions). Secondary FRET Reaction MixReaction Components 1× Volume _× Volume Single Assay Format RNASecondary Buffer 1 2.0 μl _Secondary Oligos 1.5 μl T₁₀e_(0.1) Buffer 1.5μl Total Mix Volume (1×) 5.0 μl Biplex Assay Format RNA Secondary Buffer1 2.0 μl _Secondary Oligos 1.5 μl _Housekeeping Secondary 1.5 μl TotalMix Volume (1×) 5.0 μl

[1343] 6. After the primary reaction incubation is completed, remove themicroplate seal, and add 5 μL Secondary FRET Reaction Mix per well usinga multichannel pipet. Mix by pipetting up and down 1-2 times. Reseal themicroplate with the well tape and incubate the microplate at 60° C. for60 or 90 minutes, as indicated in each Product Information Sheet. Thesecondary reaction incubation time can be varied. See sections 2 of thePROCEDURAL NOTES FOR OPERATION OF THE mRNA INVADER ASSAY for details.

[1344] 7. Reactions can be read using one of two procedures: Direct Reador Stop and Transfer.

[1345] NOTE: Remove the microplate seal before reading the microplate.

[1346] Direct Read Procedure

[1347] This procedure enables collection of multiple data sets to extendthe assay's dynamic range. During the secondary INVADER reaction, readthe microplate directly in a top-reading fluorescence microplate reader.

[1348] Recommended settings for a PerSeptive Biosystem Cytofluor 4000instrument are as follows: Specific Gene Signal Housekeeping Gene SignalExcitation 485/20 nm Excitation 560/20 nm Emission 530/25 nm Emission620/40 nm Reads/Well 10 Reads/Well 10 Gain 40 Gain 45 Temperature 25° C.Temperature 25° C. #microplate reader may need to be adjusted accordingto the manufacturer's recommendations.

[1349] Stop and Transfer Procedure

[1350] 1. Prepare 1×Stop Solution (10 mM Tris.HCl, pH 8, 10 mM EDTA)with RNase-free H₂O. Add 100 μl per well with a multichannel pipet.

[1351] 2. Transfer 1 00 μl of the diluted reactions to a blackmicroplate (e.g., COSTAR (Corning), Cat. No. 3915).

[1352] 3. Read the microplate using the same parameters as the DirectRead Procedure, but adjust the gain to give No Target Control samplereadings of 100 RFUs (see NOTE above).

[1353] In some embodiments, supplemenatary documentation, such asprotocols for ancillary procedures, e.g., for the preparation ofadditional reagents, or for preparation of samples for use in themethods of the present invention, are provided. In preferredembodiments, supplementary documentation includes guidelines and listsof precautions provided to facilitate successful use of the methods andkits by unskilled or inexperienced users. In particularly preferredembodiments, supplementary documentation includes a troubleshootingguide, e.g., a guide describing possible problems that may beencountered by users, and providing suggested solutions or correctionsto intended to aid the user in resolving or avoiding such problems.

[1354] For example, and without intending to limit such supplementarydocumentation to any particular content, kits of the present inventionmay comprise any of the following procedures and guidelines:

[1355] II. Avoidance of RNase Contamination

[1356] To avoid RNase contamination during sample preparation andtesting, in one embodiment, the user is cautioned to observe thefollowing precautions:

[1357] Wear disposable gloves at all times to avoid contact with samplesand reagents.

[1358] Use certified RNase-free disposables, including thin-wallpolypropylene tubes and aerosol-barrier pipet tips, for preparingsamples and assay reagents, to avoid cross-contamination.

[1359] Use RNase-free (DEPC-treated) H₂O for diluting samples and/orreagents.

[1360] Keep RNA samples and controls on ice during assay setup.

[1361] III. Sample and Control Preparation

[1362] NOTE: Dilute both standards and samples to concentrations thatcorrespond to a 5-μl addition per reaction.

[1363] Example 1: The concentration of a 5-attomole standard is 1amol/μl. 1 amol 10⁻¹⁸ mole=602,000 molecules.

[1364] Example 2: The concentration of a 100-ng sample should be 20ng/μl.

[1365] A. Control Preparation

[1366] No Target Control:

[1367] Total RNA Format: tRNA Carrier (20 ng/μl)

[1368] Cell Lysate Format: 1×Cell Lysis Buffer 1 (dilute 10×Cell LysisBuffer 1 to 1×with RNase-free H₂O)

[1369] Positive Control: RNA Standard (Std) (100 amol/μl in vitrotranscript)

[1370] 1. Prepare RNA standards by diluting the positive controls withtRNA Carrier (when running total RNA samples) or with 1×Cell LysisBuffer 1 [10×Cell Lysis Buffer 1 diluted with RNase-free H₂O] (whenrunning cell lysate samples). The Product Information Sheet included ineach kit indicates the recommended standard test levels and preparationmethods.

[1371] 2. Using a fresh set of standards for each run is recommended.Store the standards on ice during reaction setup.

[1372] B. Total RNA Sample Preparation

[1373] 1. Prepare total RNA from cells or tissue according tomanufacturer's instructions for the selected preparation method.Recommended methods include TRIZOL (Life Technologies, Rockville, Md.),RNEASY (Qiagen, Valencia, Calif.), and RNA WIZ (Ambion, Austin, Tex.).

[1374] 2. Dilute total RNA samples with RNase-free H₂O to theappropriate concentration.

[1375] C. Cell Lysate Sample Preparation—96-Well Microplate Format

[1376] NOTE: This cell lysate detection format is used for adherentcells cultured in 96-well tissue culture microplates. Cells aretypically seeded at 10,000-40,000 cells per well. Different seedingdensities may be required depending on cell type and/or mRNA expressionlevels. See Procedural Notes for more details. For cells exhibiting highexpression, the following methods can be used to attenuate the signalfrom the cell lysates:

[1377] plate fewer cells per well;

[1378] dilute the cell lysates with 1×Cell Lysis Buffer 1 beforeaddition to the reaction (e.g., 2.5 μl lysate+2.5 μl 1×Cell Lysis Buffer1);

[1379] read the reaction microplate 15-30 minutes after addition of theSecondary FRET Reaction Mix instead of the recommended 60-90 minutes;

[1380] 1. Dilute 10×Cell Lysis Buffer 1 to a 1×concentration withRNase-free H₂O.

[1381] 2. Using a multichannel pipet, carefully remove the culturemedium from the wells of adherent cells without disturbing the cellmonolayer.

[1382] 3. Wash the cells once with 200 μl PBS (no MgCl₂, no CaCl₂) andcarefully remove the residual PBS with the multichannel pipet.

[1383] 4. Add 40 μl 1×Cell Lysis Buffer 1 per well. Lyse cells at roomtemperature for 3-5 minutes.

[1384] 5. Using a multichannel pipet, carefully transfer 25 μl of eachlysate sample into a 96-well microplate. Avoid transferring cellularmaterial from the bottom of the well.

[1385] 6. Overlay each lysate sample with 10 μl clear CHILLOUT ormineral oil (overlaying is not necessary if using a heated-lid thermalcycler).

[1386] 7. Seal microplate with Thermaseal well tape. Immediately heatlysates at 75-80° C. for 15 minutes in a thermal cycler or oven toinactivate cellular nucleases.

[1387] 8. During the heating step, proceed with the reaction setup. SeeDETAILED mRNA INVADER ASSAY PROTOCOL (above) for instructions.

[1388] 9. After the heat inactivation step, add the lysate samplesimmediately to the reaction microplate. Alternatively, the lysatesamples can be quickly transferred to a −70° C. freezer for latertesting (long-term stability has not been established and may differ foreach cell type).

[1389] IV. Procedural Notes for Operation of The mRNA Invader Assay

[1390] 1. RNA sample types and optimization of RNA sample amount.

[1391] The assay is optimized for performance with total RNA samplesprepared from either tissue or cells. Several total RNA preparationmethods/kits have been validated for performance in the mRNA INVADERassay:

[1392] TRIZOL (Life Technologies, Rockville, Md.)

[1393] RNeasy (Qiagen, Valencia, Calif.)

[1394] RNA WIZ (Ambion, Austin, Tex.)

[1395] It is important to use a method or kit that minimizes the levelof genomic DNA, which can inhibit signal generation. Performance of apreliminary experiment is recommended to determine the amount of totalRNA sample (typically 1-200 ng, depending on the gene's expressionlevel) that provides the best limit of detection and dynamic range.

[1396] The assay has also been validated with lysate samples from anumber of cell types. Recommended cell densities in a 96-well tissueculture microplate are 10,000-40,000 cells per well depending on celltype and expression level of the gene of interest. Performance of apreliminary experiment is recommended for any given cell line and/orgene being monitored. Such an experiment should include different celldensity levels and/or dilution of the lysate samples with 1×Cell LysisBuffer 1 (e.g. a 1 μl test level is prepared by mixing 1 μl lysatesample+4 μl 1× Cell Lysis Buffer 1 for a 5 μl sample addition).

[1397] 2. Dynamic Range Modulation: Variable Secondary ReactionIncubation Times.

[1398] The length of the secondary reaction incubation time listed inthe protocol is sufficient for most analytes. However, the lineardetection range (Signal/Background<15-25) can be adjusted by reading thereaction microplate at variable times after addition of the secondaryFRET reagents. For example, high expression samples can often bedetected in 15-30 minutes. The Direct Read method (DETAILED mRNA INVADERASSAY PROTOCOL, step 7) enables simple optimization of the secondaryreaction time as the reaction microplate can be incubated further if anearly time read does not provide enough signal from the samples beingtested.

[1399] Monitoring the secondary reaction fluorescence signal with timecan also extend the dynamic range of the assay. The Direct Read methodat multiple time points can be applied using low-cost instrumentation.Alternatively, real-time fluorescence instrumentation can be used toachieve comparable dynamic ranges exhibited by other mRNA quantitationmethods.

[1400] 3. Dynamic Range Modulation: Variable Sample Levels.

[1401] While the FRET detection method greatly simplifies the assay, thedynamic range is typically limited to 2-3 logs when using an endpointread method. However, since mRNA INVADER assay signal is generatedlinearly with both target level and time, the easiest method forextending the dynamic range beyond 3 logs (as may be required, e.g., forhighly induced genes) is to adjust total RNA sample levels. Fold changesin gene expression (treated sample signal divided by untreated samplesignal) can be reliably calculated using normalized sample signals. Thisis accomplished by testing sample levels that give signal within thelinear detection range defined by the standard curve. For example, thefold induction for a highly induced sample can be calculated as follows:Fold induction=(Net Signal for 1 ng treated sample×100)/Net Signal for100 ng untreated sample

[1402] V. Troubleshooting Guide Problem Possible Solution No signalCheck that the fluorescence microplate reader has been set up correctlyand that the appropriate excitation and emission filters are in place.Perform mRNA INVADER assay with the provided standard as a positivecontrol. Potential RNase contamination of the samples and reagents.Discard suspect reagents. Use only reagents and oligonucleotidessupplied in the kit. Do not mix reagents or oligonucleotides betweenkits. High variation Always work with master primary and secondaryreaction mixes. between Thoroughly mix all master mixes and samples.replicates Pipet in a similar manner across all the controls andsamples. Calibrate pipets frequently. Lack of low Calibrate thermalcycler or heat block. target level Minimize assay variability (seeabove), i.e. CVs are less than 5% detection for the sample replicates.This is particularly important for detecting low target levels. Lack ofDecrease secondary reaction incubation time to achieve detectiondiscrimination within the linear range of the assay. between high Useless total RNA per reaction. signal samples Attenuate cell lysate samplesignal (see NOTE, Sample and Control Preparation, Part C). Signal Runsamples on an agarose gel to check for presence of genomic inhibitionDNA. Alter the RNA sample isolation method to minimize genomic DNA orpresence of other inhibitors. The same isolation procedure should beused throughout an experiment. If using the cell lysate format, residualPBS can be inhibitory. Be sure to remove residual PBS from the tissueculture microplate. Do not use PBS that contains MgCl₂ or CaCl₂, whichinhibits the assay.

[1403]

Example 20 Additional RNA INVADER Assay Embodiments

[1404] 1. ARRESTOR oligonucleotides in single-step INVADER assays.

[1405] ARRESTOR oligonucleotides can be used to selectively stop signalaccumulation for particular target nucleic acids in a reaction, suchthat signal can continue to accumulate for other target nucleic acids inthe same reaction mixture. The following is a description of oneembodiment in which it is desirable to selectively arrest a particularreaction within a mixture.

[1406] The 18S ribosomal RNA is useful as an internal standard in RNAquantitation assays, e.g., for use in normalizing the signal generatedfrom a specific gene or RNA of interest in the same sample. As aninternal control, it is preferred that the signal should report bothsample variability (e.g., dead cells in cell lysate samples, cellplating errors, RNA degradation in a particular total RNA sample) andassay variability (e.g., pipetting errors, uneven heating across theplate). Because 18S rRNA is present in samples such as cell lysates inrelatively high concentrations compared, e.g., to most mRNAs, the 18Scan often be detected in a single-step INVADER assay, without the needfor a secondary or subsequent reaction. One consideration of running asingle-step INVADER assay reaction in the same mixture as a multi-stepINVADER assay reaction is that the multi-step reactions may involvechanges of conditions (e.g., a solution addition or a temperature shift)between a primary reaction and subsequent reaction steps. For example,in the mRNA detection assays described herein, the primary reactionconditions are optimal for RNA detection, and the secondary reactionconditions are optimal for DNA detection. Accumulating single-step assaysignal under two different conditions may complicate quantitation.Further, because of the high concentration of the 18S RNA, too muchsignal may sccumulate from this target. The reaction components may beexhausted, such that the signal reaches a plateau that is not anaccurate reflection of the 18S concentration, or the accumulated signalmay overwhelm the signal from other nucleic acids in the reaction. Insome embodiments, it is preferable to limit the signal from the 18SrRNA. For example, a primary reaction with may be initiated with probesets for the 18S rRNA and for one or more other targets. An ARRESTORoligonucleotide to stop the 18S detection reaction may be added alongwith the secondary reaction reagents for the other target.

[1407] 2. Reactions without Arrestor oligonucleotides.

[1408] In some embodiments, this format involves running ahigh-temperature primary reaction (e.g. 65 C.) and a low-temperaturesecondary reaction (e.g. 55 C.) (e.g., FIG. 50A). All INVADER reactionreagents are combined at the same time, and temperature is used tosegregate the primary and secondary reactions. In this embodiment, FRETbackground does not form in the primary reaction because the FREToligonucleotide cannot stably bind to the secondary reaction template(SRT). In one embodiment of this format, the reaction components andconditions are as follows:

[1409] Reaction components

[1410] 5-10 pmoles Probe

[1411] 5 pmoles INVADER oligo

[1412] 3 pmoles Stacking oligo

[1413] 10 pmoles FRET oligo

[1414] 6-12 pmoles SRT (1.2-fold excess over Probe level)

[1415] standard RNA reaction buffer

[1416] standard MgSO4 level

[1417] 20 ng CLEAVASE enzyme (e.g., CLEAVASE VII, IX).

[1418] In preferred embodiments, primary reaction components are mixedand aliquoted into the reaction plate (e.g. 5 μl per well).

[1419] The RNA sample is added (e.g. in a 5 μl aliquot).

[1420] The reaction plate is incubated for 90 minutes (can be shorter orlonger) at 65 C. for the primary reaction.

[1421] The reaction plate is incubated for 15-90 minutes (depending onthe expected expression level and assay sensitivity) at 55 C. for thesecondary reaction.

[1422] The plate (96-well or 384-well) is measured in a fluorescenceplate reader.

[1423] In another embodiment, a Mini-Arrestor is provided (FIG. 50B)This format is run similarly to the No Arrestor format except that aMini-Arrestor oligonucleotide is included as well. In some embodiments,Mini-Arrestor oligonucleotides may be synthesized with 2′-OMenucleotides. When the temperature is reduced to start the secondaryreaction, the remaining uncleaved Probe binds to the Mini-Arrestorinstead of the SRT. Consequently, a FRET oligonucleotide in thesecondary reaction binds only to the specific secondary substrate(comprising, e.g., cleaved 5′ arm stably bound to an SRT). TABLE 2 KcatKm (dNTP) Kd (nM) Klenow (s⁻¹) (μM) affinity Relative DNA Reference TaqPol Wild-Type 2.4 2.8 8 1 2 Wild-Type S610A n.d. — n.d. — 5 S515 R668A0.006 6.5 140, 150 0.06, 0.05 1,2 R573* N678A n.d. — n.d. — 5 N583 E710A0.1 15 250 0.03 2 E615* E710D 1.7 7.7 110 0.07 2 E615* K758A 0.131 15.6— 0.63 4 K663 K758R 2.0 2.1 — 1.125 4 K663 Y766S 0.8 6.4 13 0.4, 0.6 1,2Y671 R841A 0.3 9.8 40,53 0.2 1,2 R746* N845A 1.0 23 8, 5 1.0, 1.7 1,2N750* N845Q 0.03 1.7 80, 55 0.1, 0.2 1,2 N750* Q849A 0.02 3.8 100, 1600.08, 0.05 1,2 Q754 Q849E 0.001 n.d. 90, 91 0.09 1,2 Q754 H881A 0.3 3.320, 28 0.4, 0.3 1,2 H784* D882N <0.0001 n.d. 30 0.6 2 D785 D882S 0.0017.5 0.9 9 2 D785

[1424] References:

[1425] 1. JBC (1990) 265:14579-14591

[1426] 2. JBC (1992) 267:8417-8428

[1427] 3. Eur. J. Biochem (1993) 214:59-65

[1428] 4. JBC (1994) 269:13259-13265

[1429] 5. Nature (1996) 382:278-281 TABLE 3 Rational mutations in thepolymerase region A. DNA activity table IdT % Tth % Taq 4M HP X Tth DNRX HT 31.91 100% 83% 3.81 101.9 Tth DN RX HT 23.61  74% 62% 5.32 221.24H641A Tth DN RX HT 22.1  69% 58% 4.39 88.17 R748A Tth DN RX HT 34.31108% 90% 7.75 185.35 H786A Tth DN RX HT 32.1 101% 84% 5.7 332.8H786A/G506K/ Q509K (AKK) Taq DN RX HT 38.23 120% 100%  68.21 1100.18W417L/G418K/ E507Q/H784A (Taq 4M) Taq 4M G504K 36.04 113% 94% 31.76417.40 Taq 4M H639A 42.95 135% 112%  91.46 2249.67 Taq 4M R587A 44.78140% 117%  143.0 252.69 Taq DN RX HT 43.95 138% 115%  122.53 346.56W417L/G418K/ G499R/A502K I503L/K504N/ E07K/H784A (Taq 8M) Taq SS R677A32.3 101% 84% 206.9 2450.0 B. RNA activity table % % IrT1 Tth Taq 4M TthDN RX HT 0.89 100% 34% Tth DN RX HT 1.18 133% 45% H641A Tth DN RX HT1.34 151% 51% R748A Tth DN RX HT 1.31 147% 49% H786A Tth DN RX HT 1.59179% 60% H786A/G506K/ Q509K (AKK) Taq DN RX HT 2.65 298% 100% W417L/G418K/ E507Q/H784A (Taq 4M) Taq 4M G5O4K 2.76 310% 114%  Taq 4MH639A 3.89 437% 147%  Taq 4M R587A 3.13 352% 118%  Taq DN RX HT 4.00450% 151%  W417L/G418K/ G499R/A502K/ I503L/K504N/ E07K/H784A (Taq 8M)Taq SS R677A 2.22 249% 84%

[1430] TABLE 4 Rational arch mutations DNA activity table IdT % Tth %Taq 4M HP X Taq 4M 10.20 32% 27% 2.00 97.00 P88E/P90E Taq 4M 26.30 82%69% 103.6 2900 G80E 2249.67 Taq 4M 36.45 114%  95% 19.71 749.69L109F/A110T RNA activity table % % IrT1 Tth Taq 4M Taq 4M Taq 4M 0.10 11% 4% P88E/P90E P88E/P90E Taq 4M Taq 4M 3.11 349% 117%  G80E G80E Taq4M Taq 4M 2.45 275% 92%  L109F/A110T L109F/A110T

[1431] TABLE 5 Arch/thumb combinations DNA activity table IdT % Tth %Taq 4M HP X Taq W417L/ 63.33 198% 166%  177.05 202.32 G418K/E507K/H784A/L109F/ A110T/G499R/ A502K/I503L/ G504K/E507K/ T514S (Taq SS) TaqP88E/P9OE/ 36.48 114% 95% 9.44 70.35 W417L/G418K/ G499R/A502K/I503L/G504K/ E507K/T514S/ H784A RNA activity table IrT1 % Tth % Taq 4MTaq W417L/ 3.16 355%  119% G418K/E507K/ H784A/L109F/ A110T/G499R/A502K/I503L/ G504K/E507K/ T514S (Taq SS) Taq P88E/P90E/ 0.22 25% 8%W417L/G418K/ G499R/A502K/ I503L/G504K/ E507K/T514S/ H784A

[1432] TABLE 6 Helix-hairpin-helix random mutagenesis DNA activity tableIdT % Tth % Taq 4M HP X Taq SS 23.4 73% 61% 25.7 1233.1 K198N Taq SS25.6 80% 67% 13.4 699.1 A205Q Taq SS 11.2 35% 29% 1.9 209.4 T204P Taq SS16.8 53% 44% 7.8 597.2 I200M/A205 G Taq SS 25.9 81% 68% 36.6 1429.8K203N Tth DN RX HT 10.7 33% 28% 3.2 66.3 H786A/P197R/K200R Tth DN RX HT11.5 36% 30% 6.1 327.5 H786A/K205Y Tth DN RX HT 18.3 57% 48% 2.1 98.8H786A/G203R RNA activity table IrT1 % Tth % Taq 4M Taq SS 1.22 137%  46%K198N Taq SS 0.62 70% 23% A205Q Taq SS 0.36 40% 14% T204P Taq SS 0.7787% 29% I200M/A205G Taq SS 2.09 235%  79% K203N Tth DN RX HT 0.47 52%18% H786A/P197R/K200R Tth DN RX HT 0.68 77% 26% H786A/K205Y Tth DN RX HT1.61 180%  61% H786A/G203R

[1433] TABLE 7 Random thumb mutations DNA activity table % % Taq IdT Tth4M HP X Taq DN RX HT 59.96 188% 157% 133.65 907.41 W417L/G418K/E507K/H784A/G499R/ A502K/K504N/(M1-13) Taq DN RX HT 46.74 146% 122%123.11 822.61 W417L/G418K/ /H784A/L500I/Q507H A502K/G504K (M1-36) Taq DNRX HT 85.7 269% 224% 369.96 3752.12 W417L/G418K/G499R/A502K/G504K/E507K/ H784A/T514S (M2-24) Taq DN RX HT 76.7 240% 201%355.87 2038.19 W417L/G418K/G499R/ A502K/G504K/E507K/ H784A/V518L (M2-06)RNA activity table % % Taq IrT1 Tth 4M Taq DN RX HT 2.55 287%  96%W417L/G418K/ E507K/H784A/G499R/ A502K/K504N/ (M1-13) Taq DN RX HT 2.71304% 102% W417L/G418K/ /H784A/L500I/Q507H A502K/G504K (M1-36) Taq DN RXHT 4.43 498% 167% W417L/G418K/G499R/ A502K/G504K/E507K/ H784A/T514S(M2-24) Taq DN RX HT 3.56 400% 134% W417L/G418K/G499R/A502K/G504K/E507K/ H784A/V518L (M2-06)

[1434] TABLE 8 Chimeric mutants A. DNA activity table IdT2 % Tth AKK HPX Tth AKK 34.18 100% 5 393 Taq 4M G504K 40.19 105% 28 1991 Tfi DN 2M36.60 106% 289 1326 Tsc DN 2M 25.49 75% 283 2573 Taq Tth AKK 63.89 187%32 1658 Tth Taq 4M G504K 25.03 73% 8 627 Tfi Tth AKK 34.13 100% 15 459Tsc Tth AKK 35.23 103% 29 2703 Tfi Taq 4M G5O4K 35.69 104% 37 872 TscTaq 4M 30.04 88% 25 2008 G504K B. RNA activity table IrT3 % Tth AKK TthAKK 2.27 100% Taq 4M G5O4K 2.31 102% Tfi DN 2M 0.20  9% Tsc DN 2M 0.2913% Taq Tth AKK 6.81 300% Tth Taq 4M 1.09 48% G504K Tfi Tth AKK 1.24 55%Tsc Tth AKK 9.65 4.25%  Tfi Taq 4M G504K 1.05 46% Tsc Taq 4M 2.95 130%G504K

[1435] In FIGS. 47 and 49, and throughout the specification, the terms“Z21”, “Z28” and “EQ” refer to ECLIPSE Quencher dye. “Red” and “RR”denote the REDMOND RED dye. “Epoch Yellow” refers to YAKIMA YELLOW dye.All of these dyes and quenchers are available from Synthetic Genetics,San Diego, Calif.

[1436] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described methods and systems of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the relevant fields are intended to be within the scopeof the following claims.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20030186238). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

We claim:
 1. A method for detecting the presence of a target nucleicacid comprising: a) cleaving an invasive cleavage structure, saidinvasive cleavage structure comprising an RNA target nucleic acid; andb) detecting the cleavage of said invasive cleavage structure.
 2. Themethod of claim 1, wherein cleaving is carried out by a cleavage agent.3. The method of claim 1, wherein said target nucleic acid comprises afirst region and a second region, said second region downstream of andcontiguous to said first region.
 4. The method of claim 3, wherein saidinvasive cleavage structure comprises said target nucleic acid, a firstoligonucleotide, and a second oligonucleotide, wherein at least aportion of said first oligonucleotide is completely complementary tosaid first region of said first target nucleic acid, and wherein saidsecond oligonucleotide comprises a 3′ portion and a 5′ portion, whereinsaid 5′ portion is completely complementary to said second region ofsaid target nucleic acid.
 5. The method of claim 4, wherein at leastsaid portion of said first oligonucleotide is annealed to said firstregion of said target nucleic acid and wherein at least said 5′ portionof said second oligonucleotide is annealed to said second region of saidtarget nucleic.
 6. The method of claim 1, wherein said cleavinggenerates a non-target cleavage product.
 7. The method of claim 6,wherein said detecting the cleavage of said invasive cleavage structurecomprises detecting said non-target cleavage product.
 8. The method ofclaim 4, wherein said 3′ portion of said second oligonucleotidecomprises a 3′ terminal nucleotide not complementary to said targetnucleic acid.
 9. The method of claim 4, wherein said 3′ portion of saidsecond oligonucleotide consists of a single nucleotide not complementaryto said target nucleic acid.
 10. The method of claim 1, wherein saiddetecting the cleavage of said invasive cleavage structure comprisesdetection of fluorescence.
 11. The method of claim 1, wherein saiddetecting the cleavage of said invasive cleavage structure comprisesdetection of mass.
 12. The method of claim 1, wherein said detecting thecleavage of said invasive cleavage structure comprises detection offluorescence energy transfer.
 13. The method of claim 1, wherein saiddetecting the cleavage of said cleavage structure comprises detectionselected from the group consisting of detection of radioactivity,luminescence, phosphorescence, fluorescence polarization, and charge.14. The method of claim 4, wherein said first oligonucleotide isattached to a solid support.
 15. The method of claim 4, wherein saidsecond oligonucleotide is attached to a solid support.
 16. The method ofclaim 2, wherein said cleavage agent comprises a structure-specificnuclease.
 17. The method of claim 16, wherein said structure-specificnuclease comprises a thermostable structure-specific nuclease.
 18. Themethod of claim 2, wherein said cleavage agent comprises an enzyme,wherein said enzyme comprises a heterologous functional domain, whereinsaid heterologous functional domain provides altered functionality in anucleic acid cleavage assay.
 19. The method of claim 18, wherein saidenzyme comprises a 5′ nuclease.
 20. The method of claim 19, wherein said5′ nuclease comprises a thermostable 5′ nuclease.
 21. The method ofclaim 18, wherein said enzyme comprises a polymerase.
 22. The method ofclaim 21, wherein said polymerase is altered in sequence relative to anaturally occurring sequence of a polymerase such that it exhibitsreduced DNA synthetic activity from that of the naturally occurringpolymerase.
 23. The method of claim 21, wherein said polymerasecomprises a thermostable polymerase.
 24. The method of claim 23, whereinsaid thermostable polymerase comprises a polymerase from a Thermusspecies.
 25. The method of claim 23, wherein said Thermus species isselected from Thermus aquaticus, Thermus flavus, Thermus thermophilus,Thermus filiformus, and Thermus scotoductus.
 26. The method of claim 18,wherein said heterologous functional domain comprises an amino acidsequence that provides an improved nuclease activity in said nucleicacid cleavage assay.
 27. The method of claim 18, wherein saidheterologous functional domain comprises an amino acid sequence thatprovides an improved substrate binding activity in said nucleic acidcleavage assay.
 28. The method of claim 18, wherein said heterologousfunctional domain comprises an amino acid sequence that providesimproved substrate specificity in said nucleic acid cleavage assay. 29.The method of claim 18, wherein said heterologous functional domaincomprises two or more amino acids from a polymerase domain of apolymerase.
 30. The method of claim 29, wherein at least one of said twoor more amino acids is from a palm region of said polymerase domain. 31.The method of claim 29, wherein at least one of said two or more aminoacids is from a thumb region of said polymerase domain.
 32. The methodof claim 29, wherein said polymerase comprises Thermus thermophiluspolymerase.
 33. The method of claim 29, wherein said two or more aminoacids from said polymerase domain comprise two or more amino acids fromamino acids 300-650 of SEQ ID NO:1.
 34. The method of claim 29, whereinsaid enzyme comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs:2641-2674, 2710, 2712, 2714, 2717, 2719, 2722,2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746,2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2766, 2768, 2770, 2772,2774, 2776, 2778, 2780, 2781, 2782, 2783, 2785, 2787, 2789, 2791, 2793,2795, 2797, 2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817,2819, 2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835, and
 2837. 35. Themethod of claim 29, wherein said enzyme is encoded by a nucleic acidselected from the group consisting of SEQ ID NOs:2675-2709, 2711, 2713,2715, 2716, 2718, 2720, 2721, 2723, 2725, 2727, 2729, 2731, 2733, 2735,2737, 2739, 2741, 2743 2745, 2747, 2749, 2751, 2753, 2755, 2757, 2759,2761, 2763, 2764, 2765, 2767, 2769, 2771, 2773, 2775, 2777, 2779, 2784,2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808,2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832,2834, and
 2836. 36. The method of claim 8, further comprising the stepsof forming a second invasive cleavage structure comprising saidnon-target cleavage product and cleaving said second invasive cleavagestructure.
 37. The method of claim 36, wherein said invasive cleavagestructure or said second invasive cleavage structure comprises anoligonucleotide comprising a sequence selected from the group consistingof SEQ ID NO:2837-4004.
 38. The method of claim 37, wherein saidcleavage agent comprises enzyme having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:2641-2674, 2710, 2712, 2714,2717, 2719, 2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740,2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2766,2768, 2770, 2772, 2774, 2776, 2778, 2780, 2781, 2782, 2783, 2785, 2787,2789, 2791, 2793, 2795, 2797, 2799, 2801, 2803, 2805, 2807, 2809, 2811,2813, 2815, 2817, 2819, 2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835,and
 2837. 39. The method of claim 1, wherein said RNA target nucleicacid comprises a cytochrome P450 RNA.
 40. The method of claim 1, whereinsaid RNA target nucleic acid comprises a cytokine RNA.
 41. The method ofclaim 3, wherein a portion of said target nucleic acid consisting ofsaid first region and said second region of said target nucleic acidcomprises a splice junction.
 42. The method of claim 3, wherein saidfirst region or said second region of said target nucleic acid comprisesan exon.
 43. The method of claim 3, wherein said first region or saidsecond region of said target nucleic acid comprises an intron.
 44. Themethod of claim 1, wherein said RNA target nucleic acid is provided in acell lysate.
 45. The method of claim 6, wherein said firstoligonucleotide is covalently attached to said second oligonucleotide.46. A kit for performing an invasive cleavage assay comprising a probeoligonucleotide and an invasive oligonucleotide configured to form aninvasive cleavage structure in the presence of an RNA target nucleicacid.
 47. The kit of claim 46, further comprising a polypeptide encodedby a nucleic acid selected from the group consisting of SEQ IDNOs:2675-2709, 2711, 2713, 2715, 2716, 2718, 2720, 2721, 2723, 2725,2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 2743 2745, 2747, 2749,2751, 2753, 2755, 2757, 2759, 2761, 2763, 2764, 2765, 2767, 2769, 2771,2773, 2775, 2777, 2779, 2784, 2786, 2788, 2790, 2792, 2794, 2796, 2798,2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816, 2818, 2820, 2822,2824, 2826, 2828, 2830, 2832, 2834, and
 2836. 48. The kit of claim 47,wherein said polypeptide is selected from the group consisting of SEQ IDNOs:2641-2674, 2710, 2712, 2714, 2717, 2719, 2722, 2724, 2726, 2728,2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750, 2752,2754, 2756, 2758, 2760, 2762, 2766, 2768, 2770, 2772, 2774, 2776, 2778,2780, 2781, 2782, 2783, 2785, 2787, 2789, 2791, 2793, 2795, 2797, 2799,2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819, 2821, 2823,2825, 2827, 2829, 2831, 2833, 2835, and
 2837. 49. The kit of claim 46,wherein said kit comprises an oligonucleotide comprising a sequenceselected from the group consisting of SEQ ID NO:2837-4004.
 50. The kitof claim 47, wherein said kit comprises an oligonucleotide comprising asequence selected from the group consisting of SEQ ID NO:169-21 1 and619-706, 709-2640 and 2837-4004.
 51. A method for detecting the presenceof two or more target nucleic acid sequences comprising: a) cleaving twoor more invasive cleavage structures, each of said two or more invasivecleavage structures comprising an RNA target nucleic acid having atarget sequence, wherein each of said invasive cleavage structurescomprises a different RNA target sequence; and b) detecting the cleavageof said invasive cleavage structure.
 52. A composition comprising anucleic acid sequence selected from the group consisting of SEQ IDNOs:2675-2709, 2711, 2713, 2715, 2716, 2718, 2720, 2721, 2723, 2725,2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 2743 2745, 2747, 2749,2751, 2753, 2755, 2757, 2759, 2761, 2763, 2764, 2765, 2767, 2769, 2771,2773, 2775, 2777, 2779, 2784, 2786, 2788, 2790, 2792, 2794, 2796, 2798,2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816, 2818, 2820, 2822,2824, 2826, 2828, 2830, 2832, 2834, and 2836 and the complementsthereof.
 53. A vector comprising the nucleic acid sequence of claim 52.54. A host cell comprising the vector of claim
 53. 55. A compositioncomprising a polypeptide encoded by a nucleic acid selected from thegroup consisting of SEQ ID NOs:2675-2709, 2711, 2713, 2715, 2716, 2718,2720, 2721, 2723, 2725, 2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741,2743 2745, 2747, 2749, 2751, 2753, 2755, 2757, 2759, 2761, 2763, 2764,2765, 2767, 2769, 2771, 2773, 2775, 2777, 2779, 2784, 2786, 2788, 2790,2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814,2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834, and 2836.56. The composition of claim 55, wherein said polypeptide is selectedfrom the group consisting of SEQ ID NOs:2641-2674, 2710, 2712, 2714,2717, 2719, 2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740,2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2766,2768, 2770, 2772, 2774, 2776, 2778, 2780, 2781, 2782, 2783, 2785, 2787,2789, 2791, 2793, 2795, 2797, 2799, 2801, 2803, 2805, 2807, 2809, 2811,2813, 2815, 2817, 2819, 2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835,and
 2837. 57. A method for producing an altered enzyme with improvedfunctionality in a nucleic acid cleavage assay comprising: a) providing:i) a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NOs:2641-2674, 2710, 2712, 2714, 2717, 2719, 2722,2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746,2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2766, 2768, 2770, 2772,2774, 2776, 2778, 2780, 2781, 2782, 2783, 2785, 2787, 2789, 2791, 2793,2795, 2797, 2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817,2819, 2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835, and 2837; ii) anucleic acid encoding a polypeptide comprising a sequence selected fromthe group consisting of SEQ ID NOs:2675-2709, 2711, 2713, 2715, 2716,2718, 2720, 2721, 2723, 2725, 2727, 2729, 2731, 2733, 2735, 2737, 2739,2741, 2743 2745, 2747, 2749, 2751, 2753, 2755, 2757, 2759, 2761, 2763,2764, 2765, 2767, 2769, 2771, 2773, 2775, 2777, 2779, 2784, 2786, 2788,2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810, 2812,2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834, and2836; and iii) a nucleic acid test substrate; b) introducing one or moreheterologous domains into said nucleic acid to produce an alterednucleic acid encoding an altered enzyme; c) contacting said alteredenzyme and said polypeptide with said nucleic acid test substrate toproduce cleavage products; and d) comparing cleavage products producedby said altered enzyme to cleavage products produced by saidpolypeptide.